t Ipos de HerraMIentas de Manejo del paIsaje

cell delivery systems for other injection techniques have previously been demonstra- ted, including microinjection and electroporation with good success, making use of microfluidic chips for this process. In microinjection, a microfluidic flow has been utilised to “spear” cells onto a stationary microneedle for microinjection [266], and another approach uses a deformable chip with an embedded microneedle allowing cells to be “jabbed” as they flow past [267].

Contrary to the use of microfluidics in microinjection where throughputs have been scaled up through automation, in electroporation microfluidics has been utilised to scale down electroporation from dosing bulk samples to allow treatment of single cells. Several versions of electroporation chips have been demonstrated including simple flow-by electroporation Zivet al. [268] or Baoet al.’s [269] study where cells

are characterised based on their deformablity in response to the poration. Wang

et al. use a PDMS deformable valve to generate the electrical pulse required to

permeate the membrane of cells, within a fluidic channel [270]. More recently, Zhen et al. [271] demonstrated electroporation in microdroplets, where cells and

plasmid DNA were first encapsulated within droplets, before being flowed across a pair of electrodes invoking electroporation of the contents. Zhu et al. [272] utilised

hydrodynamic focusing to squeeze cells between two buffer fluids of opposing charge that in fact form the electric field gradient for poration, in parallel to localising the cells in the centre of the channel.

6.2

Microfluidics for Optical Injection

Optical transfection posed the interesting requirement to not only allow the insertion of cells into a microfluidic chip, but to also to allow retrieval of the treated cells after poration for analysis and/or culture. Culturing the cells on chip was a possibility, but was not considered a practical option at the first stages. It is apparent that cells are in fact quite dense, and sink fairly rapidly to the base of a fluid channel. Section 2.6 details the expected behaviour of particles denser than their surrounding medium and in a microfluidic environment, finding that as a rough approximation, cells could be expected to drop out of suspension at around 10 µms−1. This had

6.2. Microfluidics for Optical Injection 131

some advantages and some disadvantages, and this effect strongly influenced the design of the microfluidic system; ultimately enabling the placing of insertion and collection ports on the chip itself rather than interfacing the external tubing.

6.2.1

Cell Delivery & Collection

First iterations of the system consisted of a near identical setup to that in Section 5.6.3. This rapidly proved to be inadequate for dealing with cells. The 0.8 mm

inside diameter of the flexible Tygon tubing meant that there was practically zero flow as far as suspended cells were concerned, causing them to come to rest within the tubing and adhering before ever reaching the chip, even after the insertion of many ml’s of sample. It was noted however, that if the piping for the sample was

made vertical, cells could be made to roll down the pipe into the chip due to gravity. This observation rapidly led to the idea of moving the section of vertical tubing for cell delivery, to a vertical sample reservoir on the chip. Behind the reservoir was placed a flow channel, which was connected to the standard tubing, and to the syringe pump. This allowed cells to be dropped directly into the chip, within a few hundred micrometres of a hydrodynamic focusing section. In the same manner, a second reservoir could be included on-chip further down stream, from which a portion of fluid could be collected. The smallest containers that could be found were 500µlmicro-centrifuge tubes, which with the bottom section removed, formed

an open bottom reservoir. The click-top lid on the tube was found to massively disturb the flow when used, so laboratory Nescofilm was used with a rubber ring to seal the top of the tube after filling.

6.2.2

PDMS Chip

The microfluidic chips for this study were fabricated in polydimethylsiloxane (PDMS) using the soft lithography procedures described in Chapter 3 and detailed in Section 4.A. The PDMS chip was designed to have an inlet and outlet port for the insertion and collection of cells, and a hydrodynamic focusing region to force the cell sample into a thin (sub-cell sized) stream down the centre of the fluidic channel thereby

6.2. Microfluidics for Optical Injection 132

directing all cells through the photoporation beam. Briefly the fluidic design was composed in a CAD software package and printed to high resolution transparency (Circuit Graphics Ltd). Photolithography was used to form a mould in 70µmthick

SU8 (Microchem) resist on a silicon wafer and silanised with perfluorooctyltrichlo- rosilane (S13125, Flurochem). Two-part PDMS (Sylgard 184, Dow Corning) was mixed in a 1:10 weight ratio, degassed, poured on to the mould and partially cured at 65oC for 45 minutes. To incorporate cell injection and collection ports, it was

necessary to define a second section of PDMS on top of the first to provide sufficient support for the cell ports, as can be observed in Figure 6.2. This was formed by placing a pre-prepared PDMS template (a silanised rectangular block with 1 cm by 2 cm section removed from the centre) on top of the first layer, and infilling with PDMS 1:10 and further baking for 2 hours at 65o_{C to fully cure the structure.}

Once cured the PDMS was peeled from the mould, inlets punched (Harris Mi- cropunch) and irreversibly sealed to a type-1 coverslip (VWR International) using a hand-held plasma treater [193]. The channel dimensions were 150 µm wide by

70µm high throughout except at the pipe inlets and reservoirs, that were 1.2 mm

and 3 mm respectively. Adapted 0.5 ml micro-centrifugation tubes (cap and base removed) were later inserted into the 3 mm punched holes to act as cell injection and collection reservoirs.

Figure 6.2: Image of the PDMS chip showing micro-centrifuge tubes for cell insertion

and collection, and the two inlet pipes on the left for buffer and sample flow and a single outlet pipe on the right.

6.2. Microfluidics for Optical Injection 133

6.2.3

Fluidic Setup

Several features were incorporated into the fluidic setup to minimise fluctuations in the fluid flow in order to obtain a steady and reproducible flow of cells over the course of the experiment. Three syringe pumps (Harvard Apparatus, Pico Plus) were used in a “push-pull” configuration to generate stable hydrodynamic focusing: one to drive the sample stream, one for the buffer streams and a third as a suction pump on the outlet. Rigid Radel R (Upchurch) tubing was used to interface the chip to the syringe pumps (see Figure 6.3), providing significantly more stable flow than the Tygon R3603 flexible tubing, that was used purely for interfacing the peristaltic pump. Four-way L-junction switching valves (Upchurch) were used to connect the peristaltic and syringe pump fluid lines, allowing the chip and pipes to be flushed and filled as desired using the peristaltic pump, but also isolated to just the syringe pump lines when running the experiment. Gastight (Hamilton) 100µlsyringes were

used with the syringe pumps.

Figure 6.3: Fluidic setup required for cleaning, sterilising and flowing cells. Cleaning,

sterilising and filling of the fluidics with cell medium is conducted using the peristaltic pump. Syringe pumps are used for flowing of the cells for optical injection, and operate in a “push-pull” configuration to obtain stable fluid flow. Layout of the PDMS microfluidic chip is also shown, which matches with Figure 6.1. The injection agent (propidium iodide) is added to the Opti-MEM® _{before filling the chip and piping. [1]}

Flow rates were controlled via a Labview interface and set to 7 µlhr−1 and 70 µlhr−1 for sample and buffer pumps respectively which, depending on the confluency

of the cell sample, gave a rate of 1 cell per second, and at a velocity of 1100 ± 100

µms−1. It should be noted that the velocity of the cells is lower than the peak

fluid velocity in the channel, because the cells were situated in the lower half of the channel due to gravity. As the cells are denser than the fluid medium, this allows