Articulo 2
Articulo 2
Articulo 2
doi: 10.1002/cbin.11150
ME THO DO LOG Y
Abstract
Renal cell carcinoma (RCC) is one of the most lethal urogenital cancers and effective treatment of metastatic RCC remains
an elusive target. Cell lines enable the in vitro investigation of molecular and genetic changes leading to renal carcinogenesis
and are important for evaluating cellular drug response or toxicity. This study details a fast and easy protocol of establishing
epithelial and fibroblast cell cultures or cell lines concurrently from renal cancer nephrectomy tissue. The protocol involves
mechanical disaggregation, collagenase digestion and cell sieving for establishing epithelial cells while fibroblast cells were
grown from explants. This protocol has been modified from previous published reports with additional antibiotics and
washing steps added to eliminate microbial contamination from the surgical source. Cell characterisation was carried out
using immunofluorescence and quantitative polymerase chain reaction. Eleven stable epithelial renal tumour cell lines of
various subtypes, including rare subtypes, were established with a spontaneous immortalisation rate of 21.6% using this
protocol. Eight fibroblast cell cultures grew successfully but did not achieve spontaneous immortalisation. Cells of epithelial
origin expressed higher expressions of epithelial markers such as pan‐cytokeratin, cytokeratin 8 and E‐cadherin whereas
fibroblast cells expressed high α‐smooth muscle actin. Further mutational analysis is needed to evaluate the genetic or
molecular characteristics of the cell lines.
Cell Biol Int 43 (2019) 715–725 © 2019 International Federation for Cell Biology 715
Establishment of cell lines from nephrectomies N. Y. Yap et al.
Cell lines provide an avenue for in vitro investigation into Methods and materials
the molecular and genetic aspects of renal cancer carcinogen-
RCC tissue and normal kidney samples were collected from
esis and pre‐clinical studies for evaluating drug response or
consented patients who have undergone nephrectomy for
toxicity at a cellular level. Primary cell cultures offer the
the removal of kidney tumour. Ethical approval was
advantage of being more biologically similar to the tumour
obtained from the University of Malaya Medical Centre
while continuous cell lines are homogenous and could be sub‐
(UMMC) Ethics Committee (Ref: 848.17) and written
cultured indefinitely. Both have their advantages and are
informed consent was obtained for each patient.
integral for in vitro experimentation. At present, most
commercially available RCC cell lines are established from
Caucasians, such as ACHN, A‐498, Caki‐1, Caki‐2, 769‐P and Materials for cell line establishment
786‐O. Asians and Caucasians differ in the incidence of RCC Table 1 lists out the solutions or media, which were
and their response to targeted treatment and immunotherapy required for the establishment of cell cultures or cell lines.
(Naito et al., 2010; Lee et al., 2014; Znaor et al., 2015). Hence, Unless stated otherwise, high glucose Dulbecco’s Modified
there might be some differences in the underlying molecular Eagle’s Medium was purchased from Nacalai Tesque
mechanisms of RCC cells in Caucasians and Asians. The (Japan) and cell culture materials or supplements were
advantage of establishing cell lines from any research centre’s obtained from Gibco (USA).
population is that patient information at diagnosis, tumour
aggressiveness or patient clinical outcome of the corresponding
Tissue collection
cell lines will be known. In addition, the molecular responses of
the established cell lines will likely conform to the character- Tissue collection was carried out aseptically with the help
istics of the study population. of the urologist surgeon and pathologist. The urologist
In this study, a fast and simple method of establishing surgeon confirmed the location of the RCC lesions during
normal kidney epithelial cortex, RCC epithelial and fibroblast tissue collection and the pathologist confirmed the
cell cultures or cell lines from primary tumours or pathological diagnosis after tissue processing. RCC tissue
nephrectomy specimen collected at surgery is described, with samples were taken within the tumour region, away from
emphasis on RCC epithelial cell lines. The outcome of the cell the tumour margin to avoid contamination with normal
line establishment and methods of initial cell characterisation kidney cells. Tumour samples were collected from the
are also presented here. This protocol has been modified from viable fleshy soft part of the tumour, avoiding necrotic,
previous published reports with additional antibiotics fibrotic or haemorrhagic areas. Normal kidney samples
(Primocin and Mycozap) and washing steps added to eliminate were collected from the outer kidney cortex with macro-
yeast/mold, bacterial and mycoplasma contamination present scopically normal appearance, which was furthest away
from the surgical source (Ebert et al., 1990; Shin et al., 2000; from the tumour lesion. Tissue collection was carried out
Park et al., 2004; Perego et al., 2005; Grimwood and Masterson, within an hour of the tumour or kidney removal from the
2009; Vesey et al., 2009; Valente et al., 2011). In addition, this is patient. Figure 1 illustrates an example of a resected kidney
the first report to present the details and protocols of with RCC from which tumour tissue was taken for cell line
simultaneous establishment of RCC, normal kidney and establishment. Each tissue sample was cut in two pieces
RCC‐associated fibroblast cell cultures or cell lines from with one part for formalin fixation [formalin fixed paraffin
multiple trials/tumours. embedded (FFPE) slides] and one part for cell line
Table 1 Solutions or media used in the establishment and maintenance of cell cultures and cell lines
Solution/media Contents
Tissue collection media DMEM with high glucose 4.5 g/L and sodium pyruvate supplemented with antibiotic–
antimycotic 3 × (penicillin 300units/mL, streptomycin 300 µg/mL and amphotericin B
0.75 µg/mL)
Phosphate‐buffered saline pH 7.4 Purchased as a 10× stock solution from First Base Laboratories
Collagenase solution Collagenase type II 1 mg/mL in tissue collection media
Culture medium I DMEM with high glucose 4.5 g/L and sodium pyruvate supplemented with 10% FBS, 1×
antibiotic–antimycotic, Primocin 100 µg/mL (Invivogen, USA) and Mycozap
prophylactic 1× (Lonza, USA)
Culture medium II/Normal culture DMEM with high glucose 4.5 g/L and sodium pyruvate supplemented with 10% FBS and
medium 1× antibiotic–antimycotic 0.25% Trypsin‐EDTA solution
Cryomedia DMEM plus 10% FBS and 10% dimethyl sulfoxide
DMEM, Dulbecco’s modified Eagle’s medium; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum.
716 Cell Biol Int 43 (2019) 715–725 © 2019 International Federation for Cell Biology
N. Y. Yap et al. Establishment of cell lines from nephrectomies
Figure 1 An example of nephrectomy specimen taken before resection of tissues for cell culture or cell line establishment. The tumour
tissue for cell line establishment was taken from a tumour area clear of necrosis or haemorrhage. The section with normal kidney tissue was
distinguishable from the tumour lesion.
establishment. The RCC tissues for cell line establishment Establishment of tumour and normal epithelial cell
(sized 0.5–1.5 cm3) were placed in separate 50 mL cen- cultures or cell lines
trifuge tubes with 5 mL of ice‐cold tissue collection media.
Procedures were performed at room temperature (≈27°C)
unless stated otherwise. Tumour and normal epithelial cells
Tissue processing were processed using a similar protocol. Tissue fragments
Tissue samples were transported to the laboratory within were transferred to clean 50 mL tubes and washed twice in
an hour after collection and tissue processing was fresh cold tissue collection media by centrifuging at 300g
performed aseptically in a Class II biosafety cabinet. for 5 min. The supernatant was removed each time, and
Kidney tumour and normal tissue were processed sepa- after the second wash, approximately 5 mL of collagenase
rately using a similar protocol. Tissue samples were first solution was added to each tube. The tubes containing the
placed on a Petri dish where fat tissue and visible blood tissue fragments were agitated using a shaking incubator at
clots were dissected out. Tissue samples were then washed 37°C for 45 min‐1 h. After enzymatic digestion, the
and agitated in cold PBS pH7.4 in sterile 50 mL tubes 4–5 digested tissue from each sample was sieved through a
times to remove any remaining blood. At this point, it is 70 μm cell strainer (SPL Life Sciences, South Korea) into a
possible to store the tissue in culture medium I at 4°C clean 50 mL tube to remove undigested tissue and
overnight (≈8–12 h) and continue processing the next day. glomeruli. The 50 mL tubes containing the cells were
However, it is advisable to proceed to the next steps centrifuged for 5 min at 300g and the supernatant was
immediately if time allows. pipetted off. The cells were re‐suspended in pre‐warmed
Tissue samples were placed on 60 mm Petri dishes and culture medium I and transferred to 25 cm2 culture flasks
minced into 1 mm3 pieces with sterile blades. Next, tissue (SPL Life Sciences). Typically, cells from each sample were
for establishment of epithelial cells was digested with seeded in two 25 cm2 flasks. Cell viability was not
collagenase (Establishment of epithelial cell lines). determined at this stage and seeding density was not
However, for fibroblast cell establishment, the minced tightly controlled. Cells were incubated at 37°C in a
pieces were washed in PBS and placed directly in culture humidified atmosphere of 5% CO2. After an overnight
flasks for propagation, without collagenase digestion incubation (12–24 h), the culture medium in the flasks was
(Establishment of fibroblast cell lines). pipetted off along with any unattached cells and blood cells.
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Establishment of cell lines from nephrectomies N. Y. Yap et al.
Attached cells were gently washed once with pre‐warmed Sciences) was placed in each well before seeding. After
wash media and replaced with fresh culture medium I. This 24 h, the growth medium was removed and cover slips
procedure was repeated after 48 and 72 h until the flasks were washed 2× with phosphate‐buffered saline (PBS).
were free of unattached cells and debris. Subsequently, Cells grown on cover slips were fixed with 4%
culture medium was changed every 2–3 days until paraformaldehyde in PBS for 20 min at room tempera-
confluency. On day 7 onwards, culture medium II was ture (RT). The cells were then washed with PBS and
used instead of culture medium I. permeabilized with 0.1% Triton X‐100 in PBS for 10 min.
Yeast and bacterial contamination were tested using the Endogenous peroxidase activity was blocked with 3%
Cell Culture Contamination Detection Kit (Molecular hydrogen peroxide (H 2O2) in PBS for 5 min. After
Probes, Thermo Fisher, USA) and presence of mycoplasma washing with PBS, the cells were incubated with primary
was detected using the MycoFluor™ Mycoplasma Detection antibody diluted to the optimised concentration in PBS
Kit (Molecular Probes, Thermo Fisher). overnight at 4°C. The dilution for pan‐cytokeratin (PCK‐
26)(Abcam, USA) was 1:150, α‐smooth muscle actin
Establishment of cancer‐associated fibroblast (α‐SMA) (1A4) (Dako, USA) was 1:800, aquaporin‐1
cell cultures (AQP‐1) (B‐11) (Santa Cruz, USA) was 1:150 and
Fibroblast cells were grown using the tissue explant technique. Tamm–Horsfall protein (THP) (B‐2) (Santa Cruz) was
Tumour tissue minced into 1 mm3 pieces were placed in a 1:200. The cells were washed with PBS and incubated
25 cm2 culture flask and pre‐warmed culture medium I was with secondary antibody (anti‐mouse/rabbit Alexa Fluor
added before incubation at 37°C in a humidified atmosphere of 488 from Thermo Fisher, USA) (1:200 dilution) before
5% CO2. After an overnight incubation (12–24 h), the culture cover slips were mounted on slides using Vectashield
medium in the flasks was pipetted off along with any aqueous mounting medium with 4′,6‐diamidino‐2‐phe-
unattached cells and debris. Attached tissue pieces were gently nylindole.
washed once with pre‐warmed wash media and replaced with Immunohistochemistry staining was also carried out using
fresh culture medium I. This procedure was repeated after 48 pan‐cytokeratin (Abcam, USA) (1:200 dilution), α‐SMA
and 72 h until the flasks were free of unattached cells and (1:400 dilution), AQP‐1 (1:150) and THP (1:150) in FFPE
debris. On day 7 onwards, culture medium II was used instead normal kidney and clear cell renal cell carcinoma (ccRCC)
of culture medium I. Fibroblast cells typically migrate out from tissue collected from the operating theatre (results not
the explant after 5–10 days. If growth was slow, 5 ng/mL of shown). This was done to determine the localisation and
fibroblast growth factor (Merck Millipore, USA) was added to expression pattern of both antibodies in intact kidney tissue.
the culture medium to encourage fibroblast growth.
Quantitative polymerase chain reaction (qPCR)
Cell culture maintenance and subculture
The expressions of epithelial and fibroblast markers were
Cells were grown to 80–90% confluency before passaging. determined in established cell lines using qPCR. Briefly, RNA
Culture medium was removed and 1 mL of 0.25% trypsin‐ was extracted from cell lines using the Trizol reagent
ethylenediaminetetraacetic acid was added to each 25 cm2 (Invitrogen, USA) according to the manufacturer’s instructions.
flask. After 5 min incubation at 37°C, the flasks were gently The extracted RNA was evaluated for RNA concentration,
tapped to detach cells and trypsin reaction was stopped with A260/A280 and A260/230 values using Nanophotometer
the addition of 1 mL culture medium II. Cells were pelleted by (Implen, USA). Acceptable A260/280 was 1.8–2.2 and A260/
centrifuging at 200g for 5 min and re‐suspended in culture 230 was 2.0‐2.2. If not immediately used, the RNA was stored
medium II before seeding into a new 25 cm2 flask. Passaging at −80°C until analysis. Complementary DNA (cDNA)
was carried out in a ½ split ratio. For cryopreservation, cells conversion was achieved using the RevertAid First Strand
were re‐suspended in 1 mL cryomedia, frozen at −80°C cDNA synthesis kit (Thermo Fisher) according to the
overnight and stored at −190°C in a liquid nitrogen tank. manufacturer’s instructions.
Cells were reactivated from cryopreservation by thawing at qPCR reaction was performed with the EvaGreen qPCR
37°C in a water bath and centrifuging the cells at 200g for Mix Plus (Solis Biodyne, Estonia). The run method used
5 min. The cells were re‐suspended in culture medium II and was as follows:
seeded into a 25 cm2 flask.
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N. Y. Yap et al. Establishment of cell lines from nephrectomies
Table 2 Primer sequences for quantitative polymerase chain reaction of epithelial and fibroblast marker expressions in cell cultures or
cell lines
Patient characteristics
UMRCC2 M 38 2a 0 0 3 ccRCC
UMRCC3 F 55 2b 0 0 2 ccRCC
UMRCC4 F 67 2a 0 0 2 ccRCC
UMRCC5 M 42 2b 1 0 4 ccRCC with sarcomatoid transformation
UMRCC6 M 67 4 1 1 4 ccRCC with sarcomatoid transformation
UMRCC7 M 69 3a 0 1 2 ccRCC
Ewing1 M 32 2b 0 0 – Ewing’s sarcoma
UMRCC8 M 64 1b 0 1 1 ccRCC
UMRCC9 F 62 1b 0 1 1 ccRCC
UMRCC10 M 62 2b 0 1 4 Mostly undifferentiated RCC with some papillary features
All immortalised cell lines were maintained for more than 20 passages. ccRCC, clear cell renal cell carcinoma; F, female; M, male.
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Figure 2 Growth characteristics of culture cells. (A) Day 1: Attachment of clumps of cells onto tissue culture flask. (B) Day 5: Cells flatten out and start to
proliferate. (C) Day 14: Cells achieving confluency with lipid/glycogen granules visible in cells. This could be seen in some clear cell RCC primary cells because
this subtype is rich in lipid/glycogen. (D) Another ccRCC cell line at confluency, which exhibits different morphology from (C). (E) Dome formation is visible in
confluent normal kidney culture, which is characteristic of proximal tubule culture. (F) Long spindly elongated cells of ccRCC fibroblast. All taken at ×100
magnification except (E), which was taken at ×40 magnification. ccRCC, clear cell renal cell carcinoma; RCC, renal cell carcinoma.
Nine immortalised cell lines were from patients with stage before senescence. The rest either grew too slowly and
T2 tumours and above, whereas two were stage T1b with could not reach confluency to be passaged, were overtaken
metastasis. In total, 6/11 (54.5%) immortalised cell lines by epithelial cells or the explant did not attach well.
were from patients who had metastasis at presentation. The Compared with RCC epithelial cells, RCC fibroblasts were
cell lines were from tumours with grade 2 and above except harder to grow and contamination of fibroblast cells in
for two with grade 1 and metastasis (both T1b tumours). RCC epithelial cultures was rarely an issue.
Figure 3 displays examples of cell lines of various subtypes, Morphologically, RCC epithelial and fibroblast cells were
which grew successfully and their different cell morphol- distinguishable (Figure 2).
ogies under the inverted microscope.
Normal epithelial kidney cortex cells (proximal tubule Immunofluorescence characterisation of cell cultures or
cells) were relatively easy to grow and proliferated at a cell lines
faster rate than the tumour epithelial cells. After the first
RCC epithelial cells stained strongly positive for the
passage, tumour epithelial cells usually reached confluency
epithelial marker, pan‐cytokeratin and negative for the
in 3–15 days while normal epithelial kidney cortex cells
fibroblast marker, α‐SMA (Figure 4). Fibroblast cells
reached confluency in 2–5 days with a 1:2 split ratio for
stained strongly for α‐SMA and negative for pan‐cytoker-
passaging. If there was normal epithelial kidney cell
atin. Normal kidney cortex cells (proximal tubule cells)
contamination in the tumour cell culture, they would
stained positive for pan‐cytokeratin and AQP‐1 (proximal
usually be morphologically distinctive and outgrow the
tubular epithelial cell marker) (Figure 4) while staining
tumour cells at initial passages (Figure S1). Therefore, it
negative or weakly for α‐SMA and the distal tubule marker
was important to avoid the tumour margin during tissue
THP (images not shown).
collection. None of the normal kidney cortex cells achieved
spontaneous immortalisation.
Out of 18 trials of growing RCC cancer‐associated qPCR characterisation of cell cultures or cell lines
fibroblast cells, none has immortalised at the moment. qPCR analysis revealed higher epithelial marker expres-
Eight fibroblast cell cultures were successfully passaged sions in these selected examples of RCC epithelial cell
once, with one cell culture proliferating up to nine passages lines compared with fibroblast cells (Figure 5). These
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N. Y. Yap et al. Establishment of cell lines from nephrectomies
Figure 3 The morphology of cultured cells at confluency (<passage 10) and their tumour counterparts (H&E staining) of various subtypes. RCC, renal
cell carcinoma.
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Establishment of cell lines from nephrectomies N. Y. Yap et al.
Figure 4 Examples of DAPI and Alexa fluor 488 staining of established RCC epithelial, RCC fibroblast and normal kidney cortex cell cultures or cell lines.
Commercial ACHN cell line was used as a comparison. (A, C, E and G) Staining for pan‐cytokeratin; (B, D and F) staining for α‐SMA. (H) Staining for AQP‐1 in
normal kidney cortex cells. α‐SMA, α‐smooth muscle actin; AQP‐1, aquaporin‐1; DAPI, 4',6‐diamidino‐2‐phenylindole; RCC, renal cell carcinoma.
722 Cell Biol Int 43 (2019) 715–725 © 2019 International Federation for Cell Biology
N. Y. Yap et al. Establishment of cell lines from nephrectomies
Figure 5 Epithelial and fibroblast marker expressions of selected RCC epithelial cell lines (UMRCC1, UMRCC2, UMRCC6 and UMRCC10) and RCC
fibroblast cells (Fibroblast 1 and 2). Data shown are representative of triplicate experiments. The expression ratio of the genes in the newly established
cells were compared with the equivalent mRNA levels found in a representative ATCC RCC epithelial cell line (ACHN). α‐SMA, α‐smooth muscle actin;
CK8, cytokeratin 8; FAP, fibroblast activation protein; mRNA, messenger RNA; RCC, renal cell carcinoma.
epithelial cell lines were designated UMRCC1 onwards. expression of fibroblast marker α‐SMA (Goodpaster
UMRCC1, UMRCC2, UMRCC6, and UMRCC10 showed et al., 2008). Interestingly, UMRCC6, a RCC cell line
higher epithelial marker (CK8 and E‐cadherin) and lower with sarcomatoid differentiation, had higher FAP ex-
fibroblast marker (α‐SMA) expressions compared with pression compared with the other epithelial cell lines
Fibroblast 1 and 2. The RCC epithelial cell lines had evaluated. FAP is marker of activated fibroblast and is
lower FAP expression compared with the fibroblast cells also expressed by cancer‐associated fibroblasts. In
except UMRCC6, which was established from a ccRCC primary RCC tumours, FAP is expressed on stromal
tumour with sarcomatoid transformation. All cell lines fibroblasts and is shown to be associated with tumour
have mixed vimentin (fibroblast marker) expression as aggressiveness, including sarcomatoid transformation
ccRCC epithelial cells are known to express this (Errarte et al., 2016). Morphologically, UMRCC6 cells
protein. were slightly spindle‐shaped and expressed high epithe-
lial markers.
Using this protocol, kidney tumour cell lines of various
Discussion
subtypes were successfully established, including un-
Epithelial RCC cell lines were established from the common types like ccRCC with sarcomatoid transforma-
primary tumour tissue of RCC patients with a sponta- tion, largely undifferentiated RCC and Ewing’s sarcoma,
neous immortalisation rate of 21.6% using this protocol. which is a non‐RCC kidney tumour. A multilocular cystic
This was slightly higher than the 12.7% rate obtained by RCC cell culture was successfully grown for three passages,
Ebert et al. (1990), which reported the spontaneous but did not achieve spontaneous immortalisation. Most
immortalisation rate of RCC cells (Ebert et al., 1990). In commercially available cell lines are of the common
this study, immortalised cell lines were from tumours subtype, ccRCC, such as Caki‐1, Caki‐2, 769‐P and 786‐0,
with more clinically aggressive characteristics such as while the subtypes of ACHN and A‐498 cell lines were
larger tumours (stage T2 and above), higher grade (grade unclear (Brodaczewska et al., 2016). Hence, these newly
2 and above) or has metastasised. The established cell established cell lines could provide a valuable resource as in
lines were confirmed to be epithelial cells with higher vitro models for rare kidney tumour subtypes. To the
expressions of epithelial markers such as pan‐cytoker- authors’ knowledge, there are no commercial RCC with
atin, CK8 and E‐cadherin (Valente et al., 2011; sarcomatoid features or Ewing’s sarcoma of the kidney cell
Subramaniam et al., 2013). These cells also exhibit lower lines, which are easily available.
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N. Y. Yap et al. Establishment of cell lines from nephrectomies
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