2.1.1 Primary Normal Human Keratinocyte Extraction and Establishment
Primary normal human oral keratinocyte (NHOK# 1, 2, 3, 4, 5, 376, 881, 355, and 113) and normal human epidermal keratinocyte strains (NHEK# 1, 2, and 3) were established from clinically normal oral buccal or gingival tissue and normal skin respectively. Donor tissues were collected in transport medium, consisting of DMEM (Lonza, Slough UK) with 3% (v/v) penicillin/streptomycin (Gibco Invitrogen, Paisley, UK). Tissues were minced using sterile scalpel and forceps, prior to digestion in Trypsin/EDTA solution (0.5 g/L of trypsin and 0.2 g/L of EDTA•4Na in Hanks' Balanced Salt Solution without CaCl2) (Gibco Invitrogen) for 4 hours at 37oC under continuous
shaking. The digested tissues were then passed through 75 μm cell strainers (BD Falcon Oxfordshire, UK) and keratinocytes were recovered underneath in DMEM medium (Lonza, Slough UK) with 10% FBS (FirstLink, Birmingham UK). Cells were spun at 140 x g for 5 minutes and finally resuspended in complete growth FAD medium with 3% (v/v) penicillin/streptomycin and 1% (v/v) antibiotic/antimycotic (PAA Laboratories, Somerset UK). FAD medium consisted of DMEM (4.5 g/L glutamine): F12 (Ham’s F12 nutrient mixture; Gibco Invitrogen) in 3:1 ratio with 10% (v/v) FBS (FirstLink, Birmingham UK) and various mitogens (0.4 µg/ml hydrocortisone, 0.1 nM cholera toxin, 5 µg/ml transferrin, 20 pM lyothryonine, 0.18 mM adenine, 5µg/ml insulin and 10 ng/ml EGF; mitogens were either bought from Sigma Aldrich, Dorset UK, or were kindly provided by Prof. Mike Philpott from the Centre for Cutaneous Research at ICMS). All cells were plated in 6 cm dishes containing pre-plated 3T3 feeder layers (for feeder layer preparation see section 2.2.1). FAD medium was replaced 72 hours later with FAD medium containing only 1% penicillin/streptomycin and FAD medium was changed every other day thereafter. The first keratinocyte colonies became visible 4-6 days after
plating. Keratinocytes were let to grow to around 80% confluence prior to cryopreservation in freezing medium consisting of 10% (v/v) DMSO (Sigma Aldrich) 90% (v/v) FBS (FirstLink, Birmingham, UK). Keratinocyte population doublings were marked 0 at this point and all experiments were carried out with keratinocytes from the frozen stocks.
2.1.2 Primary Normal Human Keratinocyte Culture
Upon recovery from liquid nitrogen, keratinocytes were grown either in FAD medium in the presence of 3T3 feeders or in serum free media (K-SFM; Gibco Invitrogen) supplemented with 25 µg/ml of bovine pituitary extract (BPE) (Gibco Invitrogen), 0.2 ng/ml of epidermal growth factor (EGF) (Gibco Invitrogen) and either low or high concentrations of calcium (0.09 mM or 0.4 mM CaCl2 respectively) (Rheinwald et al., 2002). Keratinocytes were
passaged once they reached 60-70% confluence when grown in FAD + 3T3 feeder layer co-culture or at 40-50% confluence when grown in serum free conditions. All cells were grown at 37oC in a humidified atmosphere of 10% (serum containing media) or 5% (serum-free media) CO2/95% air. In order to
obtain a pure keratinocyte population when growing keratinocytes in a 3T3 feeder co-culture system, the 3T3 feeder layer needs to be thoroughly removed. Cells were initially washed 1x in PBS and were then incubated at 37oC for 10 minutes in the presence of versene (0.2 g/L EDTA-4Na in
phosphate-buffered saline; Gibco Invitrogen). Next, versene solution containing any detached feeders was replaced with PBS. Any remaining feeders were removed by thorough and repeated pipetting, until only keratinocyte colonies were visible under the microscope. Then, keratinocytes were tyrpsinised in the presence of Trypsin/EDTA solution (0.5 g/L of trypsin and 0.2 g/L of EDTA•4Na in Hanks' Balanced Salt Solution without CaCl2,
MgCl2 • 6H2O, and MgSO4 • 7H2O) (Gibco Invitrogen) for at least 15 minutes,
until a single cell suspension of keratinocytes was obtained. Cells were then recovered in serum containing media, spun at 140 x g for 5 minutes, and finally resuspended in FAD complete medium before plating. For keratinocytes growing in serum-free media, feeder layer free conditions, versene treatment and feeder removal steps were omitted. Keratinocytes were
typically plated at a density of 5x103/cm2 and were let to grow for an average of 5-6 days upon which time they have reached 60-80% confluence.
2.1.3 Cell Line Culture
N/TERT-1 (N/TERT) keratinocytes are derived from clinically normal foreskin tissue and are further immortalised by the addition of ectopic telomerase, followed by spontaneous downregulation/loss of p16INK4a (Dickson et al., 2000). N/TERT keratinocytes were cultured in K-SFM (Gibco Invitrogen) medium. SVpgC2a oral premalignant cell line is derived from normal oral keratinocytes that have been immortalised by ectopic expression of SV40-T antigen (Kulkarni et al., 1995). SVpgC2a keratinocytes were grown in serum free EpiLife medium (Cascade Biologics, Paisley UK) (0.04 mM CaCl2) containing human keratinocyte growth supplements (HKGS; Cascade
Biologics). Normal human telomerase-immortalised oral keratinocytes OKF6/T (Dickson et al., 2000) were grown in K-SFM medium. Oral premalignant cell lines POE9n-hTERT (Rheinwald et al., 2002), POE9n (Rheinwald et al., 2002), DOK (Chang et al., 1992; Munro et al., 1999), D19 (McGregor et al., 2002), D20 (McGregor et al., 2002) and primary head and neck cancer derived cell lines CA1 and UK1 (Harper et al., 2007), CaLH2 (Harper et al., 2007), CaLH3 (Harper et al., 2007), CaDec11, CaDec12, H357 (Prime et al., 1990), 5PT (Mackenzie, 2004), PE/CA-PJ15, and Vb6 (Harper et al., 2007), were kindly provided by Prof. Ian MacKenzie (ICMS, Centre of Cutaneous Research, Queen Mary University of London). BICR31 (Loughran et al., 1997), SCC4, SCC9, SCC15, and SCC25 (Rheinwald and Beckett, 1981) cell lines were kindly provided by Prof. Ken Parkinson (Clinical and Diagnostic Oral Sciences, Queen Mary University of London). Detailed information on the keratinocyte cell lines used herein, are displayed in Table
2.1. Normal human oral and NIH3T3 fibroblasts (kindly provided by Prof.
Ken Parkinson), Phoenix amphotropic (PhxA) (human embryonic 293T derived; supplied by Nolan Lab (Medical Centre, Stanford University Medical School, CA), and HeLa cells were all grown in DMEM (Lonza) with 10% FBS (FirstLink) and 1% penicillin/streptomycin (Gibco Invitrogen). All cells were grown at 37 oC in a humidified atmosphere of 10% (serum containing media) or 5% (serum-free media) CO2/95% air. All cell lines were cultured
and passaged according to the aforementioned conditions (see section 2.1.2) that apply for primary human keratinocytes, omitting the feeder preparation/removal steps, unless needed (SCC15).
Table 2.1: Cell lines origin and culture conditions *N/A: Not Available *GF: Growth Factors
Cell Line Origin Genetic Modification Known Abnormalities Culture Medium Cell Line References
5PT HNSCC None N/A K-SFM (Mackenzie, 2004)
BICR31 Advanced non metastasized HNSCC None CDKN2A locus deletion, p53 mutation FAD (Loughran et al., 1997) CA1 HNSCC None N/A K-SFM (Harper et al., 2007) CaDec11 HNSCC None N/A FAD - CaDec12 HNSCC None N/A FAD -
CaLH2 HNSCC None N/A FAD (Harper et al., 2007) CaLH3 HNSCC None N/A FAD (Harper et al., 2007)
D19 Erythroplakia derived keratinocytes from lateral tongue
None downregulation p16INK4a FAD
(McGregor et al., 2002) D20 Severely dysplastic oral epithelium from lateral tongue
None downregulation p16INK4a K-SFM
(McGregor et al., 2002)
DOK Dysplastic Oral Epithelium of
the tongue None
p16INK4a and p53 mutation, aneuploid K-SFM (Chang et al., 1992; Munro et al., 1999) H357 HNSCC None N/A K-SFM (Prime et al., 1990) N/TERT Newborn foreskin hTERT downregulation p16INK4a K-SFM (Dickson et al., 2000)
PE/CA-
PJ15 Human Tongue SCC None N/A FAD (Kulasekara et al., 2009) OKF6/T Normal buccal mucosa hTERT downregulation p16INK4a K-SFM et al., 2002) (Rheinwald