MATERIALS AND METHODS
Chapter 2. M aterials an d methods 2.1 Materials
2.2 Apparatus
2.2.1 General laboratory equipment
The Varifuge 3.OR centrifuge was obtained from Heraeus Instruments (UK). The Cary UV-visible and UV-1601 UV-visible spectrophotometers were purchased from Varian (Australia) and Shimadzu (Japan) respectively. The Tecan Sunrise micro- titre UV-Vis spectrophotometer was obtained from Tecan (Austria). The Micro Centaur mini-centrifuge was purchased from Sanyo Gallenkamp (UK). The AMINCO Bowman series 2 luminescence spectrophotometer was obtained from Spectronic Instruments (USA). The Avanti J-25 centrifuge. Optima’’''^ LE-80K ultracentrifuge, JA- 25.50 centrifuge rotor and SW28 centrifuge rotor were from Beckman Coulter (USA). The Potter-Elvehjem homogeniser was from Jencons Scientific (Bedfordshire, UK). PDIO gel permeation chromatography (GPC) columns were from Amersham Pharmacia Biotech (Hertfordshire, UK).
2.2.2 Tissue culture
The Neubauer haemocytometer was obtained from Weber Scientific (Sussex, UK). Serological pipettes were from Elkay (Hampshire, UK). Sterile tissue-culture 96- well and 6-well plates were obtained from Costar (New York, USA). BD Plastipak syringes were obtained from Becton Dickenson (Madrid, Spain). The Galaxy S CO2
incubator was from Wolf Labs (UK). The class II type A-B3 microbiological safety cabinet AURA B was purchased from Bio Air s.c.r.l., (Milan, Italy).
2.2.3 Fluorescence microscopy
The Flexi-dry lyophiliser was from FTS systems (New York, USA). The Spectra/Por® dialysis membrane (2,000Mw cut-off) was obtained from Spectrum Laboratories (USA). The DRAM2 fluorescence microscope was from Leica Microsystems (Cambridge, UK). The FLUOstar OPTIMA fluorescence microtitre plate reader was from BMG (Offenburg, Germany). Alugram® SIL G/UV TLC plates were
C hapter 2. M aterials a n d methods
from Macherey-Nagel (Diiren, Germany). The UVGL-58 Mineralight® UV lamp was from UYP, (California, USA).
2.2.4 Agarose gel electrophoresis
The Horizon® horizontal agarose gel electrophoresis tank and the polaroid gel documenting system were from Gibco BRL (Paisley, UK). The UV transilluminator was obtained from UYP (California, USA).
2.2.5 IP EC formation
The Malvern ZetaSizer 3 was from Malvern Instruments Ltd, (Malvern, UK).
2.2.6 SDS-PAGE
The Power Pac 300 was from Bio-Rad Laboratories (Hertfordshire, UK). The Mini PROTEAN II® gel electrophoresis tank and accessories and the gel drying system including cellulose sheets and assembly table were from Bio-Rad Laboratories (Hertfordshire, UK).
2.2.7 TEM and SEM
BEEM® capsules, copper grids and holding chambers were from Agar Scientific (Essex, UK). The Philips 201 transmission electron microscope and the Philips 208 scanning electron microscope were supplied from Philips (The Netherlands).
2.3 Methods
2.3.1 Maintenance o f cells
B16F10 cells were grown in RPMI 1640 media containing Hepes (25mM) and L-glutamine supplemented with foetal calf serum (10%). HepG2 cells were grown in MEM media supplemented with foetal calf serum (10%) and non-essential amino acids (5%). Cells were maintained in an atmosphere of 5% CO2 (v/v) in a humidified CO2
incubator and were grown in 75cm^ tissue culture-treated, cantered neck flasks with vented tops (0.2pm).
2.3.2 Passaging o f cells
All cell culture procedures were carried out in a class II laminar flow cabinet, where aseptic conditions were employed. Cells were passaged when they showed 70- 80% confluency, in order to ensure an exponential growth phase. A 75cm^ flask was
C hapter 2. M aterials and methods
washed twice in lOmL PBS (O.IM) (in order to remove cell adhesion proteins) and ImL pre-warmed trypsin (0.25%)-ETDA (ImM) was added. After a 5min incubation at 37°C, the cells became a suspension and 9mL pre-warmed media was added to the flask to dilute the trypsin. The cell suspension was then passed through a 20-gauge needle attached to a syringe to break up any cell aggregates and minimise cell clumping. ImL was removed from this flask and used to seed a fresh tissue culture 75cm^ flask containing 9mL pre-warmed media (1:10 split ratio). Flasks were then returned to the incubator and until cell growth reached 60-70% confluence or used in an experimental procedure. Peissage numbers were recorded after every cell passage, and the maximum cell passage number used was 40.
B16F10 cells were passaged twice every week. HepG2 cells were passaged once every week.
2.3.3 Evaluation o f cell number and cell viability
For quick cell counting a haemocytometer slide was used. The grid on this glass slide acts as a cell counting chamber. Cell viability can additionally be assessed using trypan blue dye. Trypan blue is only able to penetrate dead cells, thus viable cells are not stained and can be counted.
In order to prepare a Neubauer haemocytometer slide for cell counting, the edges of a precision ground coverslip were wet slightly and the coverslip pressed firmly onto the haemocytometer slide until Newton’s rings appeared. Next, a cell suspension (20|iL) was mixed with an equal volume of 2% (v/v) trypan blue dye (supplied as 4% (v/v) and diluted in PBS at a 1:1 ratio) and a drop (lOpL) was placed at the edge of the coverslip using a pipette. The slide was then placed under an inverted light microscope and all viable cells counted (dead cells were stained blue) in a quadrant of known volume (0.1 mm^). The number of viable cells was then determined by the following formula:
Total number of viable cells/mL = mean number of cells per quadrant x dilution factor
( 2 ) X 1 0 “ .
2.3.4 Freezing-down cells
A cell bank where cells were stored either frozen at -80^C or in liquid nitrogen vapour, was created to ensure that when cell passage numbers reached over 40, new viable cells of low passage number could be easily recovered and used in subsequent assays.
C hapter 2. M aterials an d methods
First, sterile freezing media made from PCS (90% (v/v)) and DMSO (10% (v/v)) was filter sterilised (0.2^im filter) and warmed to 37®C in a water-bath. Trypsinised cells diluted with media were then counted using a Neubauer haemocytometer. The number was recorded and the remaining cells transferred to a sterile centrifuge tube and spun at l,500g x 5min at 37^C. Following this, the supernatant was removed and cells were resuspended in the appropriate volume of freezing media to give a cell number of 1x10^ cells/mL and disaggregated using a 20-gauge needle. Next, ImL of the cell suspension was transferred into a sterile cryogenic vial. In order to ensure that cells were frozen slowly at a rate o f l°C/min, vials were wrapped in tissue paper, placed in a polystyrene box, placed at -20®C for Ih and then at -80°C overnight. The next morning, cells were placed in liquid nitrogen (-196®C) for long term storage.
2.3.5 Cell recovery from cell banks
To recover the cryogenically frozen cells, the lids o f the cryogenic vials were turned one-quarter and placed in a sterile 30mL universal bottle at 37°C. Once the preparation started to thaw (~ 3min), the preparation was centrifuged at l,500g x 5min at 22®C and the supernatant removed. Cells were resuspended in ImL of the appropriate cell culture media for the cell line and used to seed a 25cm^ tissue culture- treated, cantered neck flask with vented top (0.2pm) which contained 4mL of the appropriate cell culture medium. Once cells were confluent (~ 4 days), cells were passaged and seeded into 75cm^ flasks.
2.3.6 Evaluation o f cell viability using the colourimetric tetrazolium-based 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay
To ascertain the effect of potential toxicity o f polymers on cells, a cytotoxicity assay was performed. Sterile, flat-bottomed 96-well plates were seeded with B16F10 or HepG2 cells to a density of 1 x 10"^ cells/well and incubated at 37°C in an atmosphere of CO2 (5% (v/v)) for 24h. Polymers were dissolved in media to give stock solutions (0.05
and 2mg/mL) and were filter sterilised and then added to the cells to give a final concentration range of 0-2mg/mL as described in table 2.1.
After incubation for 67 h, cell viability was assessed using the MTT assay (Sgouras and Duncan, 1990). This assay is based on the ability of mitochondrial dehydrogenase enzymes in viable cells to metabolise a water-soluble tétrazolium dye (MTT) into an insoluble formazan salt as seen in figure 2.1. MTT (20pL of a 5mg/mL solution in PBS) was added to each well and the plates were subject
C hapter 2. M aterials a n d methods
Table 2.1. Polymer concentrations used in the cytotoxicity assay Polymer concentration (mg/mL) Amount polymer (pL) Amount media (pL) Stock (mg/mL) 2 100 0 2 1 50 50 2 0.5 25 75 2 0.1 5 95 2 0.05 100 0 0.05 0.01 20 80 0.05 0.005 10 90 0.05 0.001 2 98 0.05 0 0 100 0
C hapter 2. M aterials a n d methods Phe
\
/
\
Phe MTT (yellow) mitochondrial enzymes Phe N .N- Phe"/
Ny
.CH3MTT formazan salt (blue)
*CH3