Establishment of epithelial and fibroblast cell lines from primary renal cancer nephrectomies

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 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 characterization was carried out using immunofluorescence and quantitative PCR. Eleven stable epithelial renal tumour cell lines of various subtypes, including rare subtypes, were established with a spontaneous immortalization rate of 21.6% using this protocol. Eight fibroblast cell cultures grew successfully but did not achieve spontaneous immortalization. Cells of epithelial origin expressed higher expression of epithelial markers such as pan-cytokeratin, CK8 and E-cadherin whereas fibroblast cells expressed high α-SMA. Further mutational analysis is needed to evaluate the genetic or molecular characteristics of the cell lines.


Introduction
Renal cell carcinoma (RCC) comprises of 2-3% of all human malignancies and the incidence is increasing worldwide [1]. The incidence of RCC is higher in Western countries (North America, Europe, Australia and New Zealand) compared to Asian countries in general [1].
The most common subtypes of RCC are clear cell (70-80%) followed by papillary (10%), chromophobe (5%) and collecting duct RCC (1%) [2]. RCC with sarcomatoid or rhabdoid transformation is not a recognised subtype of RCC as sarcomatoid or rhabodoid features can be found in all histologic subtypes of RCC [3]. Both of these histological transformations in RCC are associated with aggressive tumours and poor prognosis.
Radiation and chemotherapy has limited efficacy on RCC. The current approved targeted therapies for metastatic RCC suppress angiogenesis by inhibiting vascular endothelial growth factor (VEGF) or platelet-derived growth factor (PDGF) mediated pathways. However, most patients eventually develop resistance to these drugs and require second or third-line therapies [4]. Immune checkpoint inhibitors are the most recent approved for treatment of metastatic RCC. Nevertheless, since these drugs are relatively new, the long term clinical outcome is unknown at the moment. Hence, the genetic and molecular changes leading to the pathogenesis of RCC and development of therapy resistance are still not well understood. Caucasians differ in the incidence of RCC and their response to targeted treatment and immunotherapy [1,5,6]. Hence, there might be some difference in the underlying molecular mechanisms of RCC cells in Caucasians and Asians. The advantage of establishing cell lines from any research centre's population is that patient information at diagnosis, tumour aggressiveness or patient clinical outcome of the corresponding cell lines will be known. In addition, the molecular responses of the established cell lines will likely conform to the characteristics of the study population.
In this study, a fast and simple method of establishing RCC epithelial and fibroblast as well as normal kidney epithelial cortex cell lines from primary tumours or nephrectomy specimen collected at surgery is described, with emphasis on RCC epithelial cell lines. The outcome of the cell line establishment and methods of initial cell characterization are also presented here.
This protocol has been modified from previous published reports with additional antibiotics (Primocin and Mycozap) and washing steps added to eliminate yeast/mold, bacterial and mycoplasma contamination present from the surgical source [7][8][9][10][11][12][13]. In addition, this is the first report to present the details and protocols of simultaneous establishment of RCC, normal kidney and RCC associated fibroblast cell lines from multiple trials/tumours. and written informed consent was obtained for each patient. was purchased from Nacalai Tesque (Japan) and cell culture materials or supplements were obtained from Gibco (USA).

Tissue collection
Tissue collection was carried out aseptically with the help of the urologist surgeon and pathologist. The urologist surgeon confirmed the location of the RCC lesions during tissue collection and the pathologist confirmed the pathological diagnosis after tissue processing.
RCC tissue samples were taken within the tumour region, away from the tumour margin.
Tumour samples were collected from the fleshy soft part of the tumour, avoiding necrotic, fibrotic or haemorrhagic areas. Normal kidney samples were collected from the outer kidney cortex with macroscopically normal appearance which was furthest away from the tumour lesion. Tissue collection was carried out within an hour of the tumour or kidney removal from the patient. were placed in separate 50ml centrifuge tubes with 5ml of ice-cold tissue collection media.

Tissue Processing
Tissue samples were transported to the laboratory within an hour after collection and tissue processing was performed aseptically in a Class II biosafety cabinet. Kidney tumour and normal tissue were processed separately using a similar protocol. Tissue samples were first placed on a petri dish where fat tissue and visible blood clots were dissected out. Tissue samples were then washed and agitated in cold PBS pH7.4 in sterile 50ml tubes 4-5 times to remove any remaining blood. At this point, it is possible to store the tissue in culture medium I at 4°C overnight (≈ 8-12 hours) and continue processing the next day. However, it is advisable to proceed to the next steps immediately if time allows.
Tissue samples were placed on 60mm petri dishes and minced into 1mm 3 pieces with sterile blades. Next, tissue for establishment of epithelial cells was digested with collagenase (Establishment of epithelial cell lines). However, for fibroblast cell establishment, the minced pieces were washed in PBS and placed directly in culture flasks for propagation, without collagenase digestion (Establishment of fibroblast cell lines).

Establishment of tumour and normal epithelial cell lines
Procedures were performed at room temperature (≈27°C) unless stated otherwise. Tumour and normal epithelial cells were processed using a similar protocol. Tissue fragments were transferred to clean 50ml tubes and washed twice in fresh cold tissue collection media by centrifuging at 300g for 5 minutes. The supernatant was removed each time, and after the second wash, approximately 5ml of collagenase solution was added to each tube. The tubes containing the tissue fragments were agitated using a shaking incubator at 37°C for 45 minutes-1 hour. After enzymatic digestion, the digested tissue from each sample was sieved through a 70μm cell strainer (SPL Life Sciences, South Korea) into a clean 50ml tube to remove undigested tissue and glomeruli. The 50ml tubes containing the cells were centrifuged for 5 minutes at 300g and the supernatant was pipetted off. The cells were resuspended in pre-warmed culture medium I and transferred to 25cm 2 culture flasks (SPL Life Sciences, South Korea). Typically, cells from each sample were seeded in two 25cm 2 flasks.
Cell viability was not determined at this stage and seeding density was not tightly controlled.
Cells are incubated at 37°C in a humidified atmosphere of 5% CO 2 . After an overnight incubation (12-

Establishment of cancer associated fibroblast cell lines
Fibroblast cells were grown using the tissue explant technique. Tumour tissue minced into 1mm 3 pieces was placed in a 25cm 2 culture flask and pre-warmed culture medium I was added before incubation at 37°C in a humidified atmosphere of 5% CO 2 . After an overnight incubation (12-24 hours), the culture medium in the flasks was pipetted off along with any unattached cells and debris. Attached tissue pieces were gently washed once with prewarmed wash media and replaced with fresh culture medium I. This procedure was repeated after 48 and 72 hours until the flasks were free of unattached cells and debris. On day 7 onwards, culture medium II was used instead of culture medium I. Fibroblast cells typically migrate out from the explant after 5-10 days. If growth was slow, 5ng/ml of fibroblast growth factor (FGF) (Merck Millipore, USA) was added to the culture medium to encourage fibroblast growth.

Cell culture maintenance and subculture
Cells were grown to 80-90% confluency before passaging. Culture medium was removed and 1ml of 0.25% trypsin-EDTA was added to each 25cm 2 flask. After 5 minutes incubation at 37°C, the flasks were gently tapped to detach cells and trypsin reaction was stopped with the addition of 1ml culture medium II. Cells were pelleted by centrifuging at 200g for 5 minutes and re-suspended in culture medium II before seeding into a new 25cm 2 flask.
Passaging was carried out in a ½ split ratio. For cryopreservation, cells were re-suspended in 1ml cryomedia, frozen at -80°C overnight and stored at -190°C in a liquid nitrogen tank.
Cells were reactivated from cryopreservation by thawing at 37°C in a water bath and centrifuging the cells at 200g for 5 min. The cells were re-suspended in culture medium II and seeded into a 25cm 2 flask.

Cell characterization by immunofluorescence staining
General cell morphology was viewed under an inverted microscope. Cells were seeded at

Quantitative PCR (qPCR)
The expression of epithelial and fibroblast markers was determined in established cell lines using qPCR. Briefly, RNA was extracted from cell lines using the Trizol reagent (Invitrogen,

Establishment of renal tumour and normal kidney cell lines
After optimizing the establishment method, 11 cell lines spontaneously immortalised out of 51 trials (from different patients). Therefore, the spontaneous immortalisation rate of tumour epithelial cells was 21.6% with the optimized protocol. Cells were considered immortalised if they could be passaged beyond the 10 th passage. Most cells senesced and stopped proliferating after 3-5 passages. Out of the spontaneously immortalised cell lines, 7 were clear cell RCC (ccRCC) variants, 2 were ccRCC with sarcomatoid transformation, one was mostly undifferentiated RCC with some papillary features and one was non-RCC Ewing's sarcoma. Each cell line had distinctive morphology ranging from polygonal epithelial to spindle shaped and elongated cells (Fig 2). Nine immortalised cell lines were from patients with stage T2 tumours and above, whereas two were stage T1b with metastasis. In total, 6/11 (54.5%) immortalised cell lines were from patients who had metastasis at presentation. The cell lines were from tumours with grade 2 and above except for two with grade 1 and metastasis (both T1b tumours).   Out of 18 trials of growing RCC cancer associated fibroblast cells, none has immortalised at the moment. Eight fibroblast cell cultures were successfully passaged once, with one cell culture proliferating up to nine passages before senescence. The rest either grew too slowly and could not reach confluency to be passaged, were overtaken by epithelial cells or the explant did not attach well. Compared to RCC epithelial cells, RCC fibroblasts were harder to grow and contamination of fibroblast cells in RCC epithelial lines was rarely an issue.

Immunofluorescence characterisation of cell lines
RCC epithelial cells stained strongly positive for the epithelial marker, pan-cytokeratin and negative for the fibroblast marker, α-SMA (Fig 4). Fibroblast cells stained strongly for α-SMA and negative for pan-cytokeratin. Normal kidney cortex cells (proximal tubule cells) stained positive for pan-cytokeratin and AQP-1 (proximal tubular epithelial cell marker) (Fig   4) while staining negative or weakly for α-SMA and the distal tubule marker THP (images not shown).

Discussion
Epithelial RCC cell lines were established from the primary tumour tissue of RCC patients with a spontaneous immortalisation rate of 21.6% using this protocol. This was slightly higher than the 12.7% rate obtained by Ebert et al. (1990), which reported the spontaneous immortalisation rate of RCC cells [9]. In this study, immortalised cell lines were from tumours with more clinically aggressive characteristics such as larger tumours (stage T2 and above), higher grade (grade 2 and above) or has metastasized. The established cell lines were confirmed to be epithelial cells with higher expressions of epithelial markers such as pancytokeratin, CK8 and E-cadherin [7,14]. These cells also exhibit lower expression of fibroblast marker α-SMA [15]. Interestingly, UMRCC6, a RCC cell line with sarcomatoid differentiation, had higher FAP expression compared to the other epithelial cell lines evaluated. FAP is marker of activated fibroblast and is also expressed by cancer associated fibroblasts. In primary RCC tumours, FAP is expressed on stromal fibroblasts and is shown to be associated with tumour aggressiveness, including sarcomatoid transformation [16].
Morphologically, UMRCC6 cells were slightly spindle shaped and expressed high epithelial markers.
Using  [7][8][9][10][11][12]. Based on the trials during this protocol optimization, the growth of the epithelial cells before the first passage for the explant method was slower compared to using enzymatic tissue digestion. Therefore, the epithelial protocol described here entails enzymatic digestion followed by cell sieving. Contamination with fibroblast cells was seldom an issue in this study as RCC tumour associated fibroblast cells were more difficult and slower to grow than the epithelial cells. Fibroblast cells were more successfully grown using the explant method, similarly described by previous groups [13,18]. Due to the limited publications on cancer associated fibroblasts in RCC, the optimization of the fibroblast establishment protocol or technique could be pursued further. This is because cancer associated fibroblasts are known to interact with cancer cells to promote tumour growth and progression [19].
Compared to previously reported protocols, additional antibiotics and washing steps were added as a precaution to prevent bacterial and mycoplasma growth as primary cultures from human tissues can suffer from contamination issues [7][8][9][10][11][12]20,21]. Using this protocol, coating of culture flasks is not required and washed tumour/normal kidney tissue can be left overnight in culture medium at 4°C before further tissue processing with good success rate.
This allows for more convenient tissue processing of specimens collected from operation cases which are carried out in the evenings or at night.

Conclusions
In summary, the protocol described in this paper allows for simultaneous establishment of RCC, normal kidney and RCC associated fibroblast cell lines or cultures from nephrectomy specimens with a good success rate of spontaneous immortalization for RCC epithelial cell lines. Tissue location selection from the surgical specimen is important and morphological, immunofluorescence and qPCR characterization can be carried out to determine the epithelial or fibroblastic origin of the cells. Further characterization via mutation analysis can be performed next to determine the genetic mutational or molecular features of the cell lines.