Name: establishment of a primary culture of patient-derived soft tissue sarcoma
Uploaded: 2018-04-11T13:00:00.000+00:00
Duration: 7 min 55 s
Description: Watch this Scientific Journal Video about Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma at JoVE.com

The authors would like to thank Gr&#225;inne Tierney for editorial assistance.

STS cells are isolated from a tumor mass surgically excised from patients with STS. The protocol has been approved by the Local Ethics Committee and is performed according to Good Clinical Practice guidelines and the Declaration of Helsinki. All patients gave written informed consent to take part in the study. The surgical specimens are analyzed by an experienced sarcoma pathologist and processed within 3 hours of surgery.
1. Tumor Specimen Collection and Processing:
<ol>
	<li>Collect tumor specimens with the help of a sarcoma pathologist in a 100-mL sterile urine container with 50 mL of DMEM low glucose medium sealed with a paraffin film.</li>
	<li>Store the samples at 4 &#176;C for a maximum of 3 h after tumor resection.</li>
	<li>Perform all the following steps under a sterile tissue culture hood.
	<ol>
		<li>Sterilize the hood with 70% ethanol and wear sterile gloves.</li>
		<li>Sterilize steel inox tweezers with 70% ethanol (use a sterile gauze).</li>
		<li>Remove the paraffin film from the urine container and discard.</li>
		<li>Wash exterior of the urine container with 70% ethanol.</li>
		<li>Open a square petri dish.</li>
		<li>Open the urine container and transfer the tumor specimens into the square petri dish using the sterilized steel inox tweezers.</li>
	</ol>
	</li>
	<li>Wash the tumor specimens with PBS twice.</li>
	<li>Open a sterile surgical scalpel.
	<ol>
		<li>Finely shred the tumor specimens into 1 - 2 mm3 pieces.</li>
		<li>Collect one quarter of the total sample in a 2-mL tube.</li>
		<li>Add 1 mL of trizol reagent (purchased) to the tube and immediately freeze at -80 &#176;C (the sample will be used in the downstream analysis for the STS gene expression evaluation).</li>
	</ol>
	</li>
	<li>Collect the rest of the finely shredded tumor material in a 15-mL tube.
	<ol>
		<li>Add 4 mL of collagenase type I solution (collagenase type I is dissolved in PBS, at a concentration of 4 &#181;g/mL).</li>
		<li>Dilute the collagenase type I solution with 4 mL of DMEM high culture media (final concentration of collagenase type I is 2 &#181;g/mL).</li>
		<li>Close the 15-mL tube and seal with paraffin film.</li>
		<li>Put the 15-mL tube in a rolling shaker in an incubator at 37 &#176;C in stirring conditions for 15 min to activate the digestion.</li>
		<li>After 15 min store the tube in the rolling shaker under stirring conditions at room temperature overnight.</li>
	</ol>
	</li>
	<li>The following day put the 15-mL tube under the sterile hood.</li>
	<li>Gently filter the cell suspension through a 100 &#181;m sterile filter attached to the top of a 50-mL tube.</li>
	<li>Wash the filter with DMEM high culture media supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin/streptomycin.</li>
	<li>Discard the 100 &#181;m sterile filter, close the 50-mL tube and centrifuge at 225 x g for 5 minutes at room temperature.</li>
	<li>Discard the supernatant by inverting the tube.</li>
	<li>Re-suspend the cells with 1 - 2 mL DMEM high culture media supplemented with 10% fetal bovine serum, 1% glutamine,&#160;and 1% penicillin/streptomycin. 
	Note: The volume depends on the amount of tumor sample processed.</li>
	<li>Count the cells with a Neubauer chamber.
	<ol>
		<li>Dilute the sample 1:2 in trypan blue and use 10 &#181;L of the dilution to count cells.</li>
		<li>Count at least twice and calculate the mean of the replicates to know the total mean number of cells collected.</li>
	</ol>
	</li>
	<li>Plate cells in a standard monolayer culture at a density of 80,000 cells/cm2. 
	Note: Always include controls in experiments. Perform at least 3 technical replicates for each condition. Perform at least 2 biological replicates. For preclinical pharmacology, gene expression and protein expression analyses, experiments should be carried out with a low number of culture passages within 30 - 45 days of cell seeding to avoid losing cancer cell phenotypes.</li>
	<li>Cytological sample preparation.
	<ol>
		<li>Collect 100,000 cells in 200 &#181;L of DMEM high culture media supplemented with 10% fetal bovine serum, 1% glutamine,&#160;and 1% penicillin/streptomycin.</li>
		<li>Pipette the solution (200 &#181;L pipette) into a disposable funnel equipped with a filter and mounted with a glass slide.</li>
		<li>Open the cytocentrifuge and insert the samples.</li>
		<li>Run the cytocentrifuge at 18.6 x g for 20 min.</li>
		<li>Discard the funnel equipped with the filter.</li>
		<li>Fix the slides in acetone for 10 min followed by chloroform for 5 min.</li>
		<li>Store the slides at -20 &#176;C. 
		Note: Thaw and hydrate the samples before staining and perform the staining following the manufacturer&#39;s instructions. Prepare at least 3 technical replicates.</li>
	</ol>
	</li>
</ol>
2. STS Cell Cultures:
<ol>
	<li>Change media once a week (DMEM high culture media supplemented with 10% fetal bovine serum, 1% glutamine and 1% penicillin/streptomycin).
	<ol>
		<li>Discard the culture medium using a 200 - 1,000 &#181;L pipette.</li>
		<li>Add new fresh medium using a 200 - 1,000 &#181;L pipette.</li>
		<li>Cell passage:
		<ol>
			<li>Detach cells when the confluence is around 90%: 
			Note: STS cell culture amplification should be performed once a week on the basis of the culture conditions.</li>
			<li>Discard medium, wash with PBS and add 2-3 mL of trypsin 1x in PBS 
			Note: The volume of trypsin used depends on the amount of cultured cells obtained.</li>
			<li>Incubate cells for 3 - 5 min at 37 &#176;C.</li>
			<li>Collect detached cells from the plate using a 200 - 1,000 &#181;L pipette.</li>
			<li>Pipette the detached cells into a 15-mL tube and add 4 mL of DMEM high culture media supplemented with 10% fetal bovine serum, 1% glutamine,&#160;and 1% penicillin/streptomycin to stop the enzymatic reaction.</li>
			<li>Centrifuge at 225 x g for 5 min at room temperature.</li>
			<li>Discard the supernatant.</li>
			<li>Re-suspend the cell pellet by adding 1 mL of DMEM high culture media supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin/streptomycin.</li>
			<li>Seed the cells in a new culture plate or a flask with a final volume according to the manufacturer&#39;s instructions. 
			Note: Do not split cells more than 1:3.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Downstream Analyses:
Note: A variety of different experiments can be carried out using this model (see below). It is, therefore, crucial to decide during the design phase of the study which analyses will be performed so that an adequate number of samples and replicates can be prepared.
<ol>
	<li>Proliferation assays such as MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide</li>
	<li>Apoptotic assays such as terminal deoxynucleotidyl transferase (TdT) nick end labeling (TUNEL) assay</li>
	<li>Immunohistochemical analysis</li>
	<li>Gene expression analysis</li>
	<li>Western blot analysis</li>
	<li>ELISA assay</li>
	<li>Fluorescence-activated cell sorting (FACS) analysis</li>
	<li>In vivo xenograft model</li>
</ol>

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J Clin. 66 (1), 7-30 (2016).</li><li>Linch, M., Miah, A. B., Thway, K., Judson, I. R., Benson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+treatment+of+soft-tissue+sarcoma+gold+standard+and+novel+therapies.">Systemic treatment of soft-tissue sarcoma gold standard and novel therapies.</a> Nat Rev Clin Oncol. 11 (4), 187-202 (2014).</li><li>Fletcher, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+classification+of+soft+tissue+tumours+-+an+update+based+on+the+new+2013+WHO+classification.">The evolving classification of soft tissue tumours - an update based on the new 2013 WHO classification.</a> Histopathology. 64 (1), 2-11 (2014).</li><li>De Vita, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+classification,+treatment+options,+and+new+perspectives+in+the+management+of+adipocytic+sarcomas.">Current classification, treatment options, and new perspectives in the management of adipocytic sarcomas.</a> Onco Targets Ther. 9 (12), 6233-6246 (2016).</li><li>Recine, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+the+role+of+trabectedin+in+the+treatment+of+intractable+soft+tissue+sarcomas.">Update on the role of trabectedin in the treatment of intractable soft tissue sarcomas.</a> Onco Targets Ther. 10, 1155-1164 (2017).</li><li>Ducimeti&#232;re, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+sarcoma+histotypes+and+molecular+subtypes+in+a+prospective+epidemiological+study+with+central+pathology+review+and+molecular+testing.">Incidence of sarcoma histotypes and molecular subtypes in a prospective epidemiological study with central pathology review and molecular testing.</a> PLoS One. 6 (8), 20294 (2011).</li><li>Nussbaum, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preoperative+or+postoperative+radiotherapy+versus+surgery+alone+for+retroperitoneal+sarcoma:+a+case-control,+propensity+score-matched+analysis+of+a+nationwide+clinical+oncology+database.">Preoperative or postoperative radiotherapy versus surgery alone for retroperitoneal sarcoma: a case-control, propensity score-matched analysis of a nationwide clinical oncology database.</a> Lancet Oncol. 17 (7), 966-975 (2016).</li><li>The ESMO/European Sarcoma Network Working Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+tissue+and+visceral+sarcomas:+ESMO+clinical+practice+guidelines+for+diagnosis,+treatment+and+follow-up.">Soft tissue and visceral sarcomas: ESMO clinical practice guidelines for diagnosis, treatment and follow-up.</a> Ann Oncol. 25, 102-112 (2014).</li><li>Paul, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cancer+Cell+in+Vitro:+A+Review.">The Cancer Cell in Vitro: A Review.</a> Cancer Research. 22, 431-440 (1962).</li><li>Morton, C. L., Houghton, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+tumor+xenografts+in+immunodeficient+mice.">Establishment of human tumor xenografts in immunodeficient mice.</a> Nat Protoc. 2 (2), 247-250 (2007).</li><li>Centenera, M. M., Raj, G. V., Knudsen, K. E., Tilley, W. D., Butler, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+culture+of+human+prostate+tissue+and+drug+development.">Ex vivo culture of human prostate tissue and drug development.</a> Nat Rev Urol. 10 (8), 483-487 (2013).</li><li>Vannucci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroma+as+an+Active+Player+in+the+Development+of+the+Tumor+Microenvironment.">Stroma as an Active Player in the Development of the Tumor Microenvironment.</a> Cancer Microenviron. 8 (3), 159-166 (2015).</li><li>De Vita, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+Eribulin+in+a+Primary+Culture+of+Well-Differentiated/Dedifferentiated+Adipocytic+Sarcoma.">Activity of Eribulin in a Primary Culture of Well-Differentiated/Dedifferentiated Adipocytic Sarcoma.</a> Molecules. 3 (12), 1662 (2016).</li><li>Failli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenge+of+culturing+human+colorectal+tumor+cells:+establishment+of+a+cell+culture+model+by+the+comparison+of+different+methodological+approaches.">The challenge of culturing human colorectal tumor cells: establishment of a cell culture model by the comparison of different methodological approaches.</a> Tumori. 95 (3), 343-347 (2009).</li><li>Steinstraesser, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+primary+human+sarcoma+model+in+athymic+nude+mice.">Establishment of a primary human sarcoma model in athymic nude mice.</a> Hum. Cell. 23, 50-57 (2010).</li><li>Raouf, A., Sun, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+methods+to+culture+primary+human+breast+epithelial+cells.">In vitro methods to culture primary human breast epithelial cells.</a> Methods Mol. Biol. 946, 363-381 (2013).</li><li>Daigeler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+in+vitro+effects+of+doxorubicin+on+gene+expression+in+primary+human+liposarcoma+cultures.">Heterogeneous in vitro effects of doxorubicin on gene expression in primary human liposarcoma cultures.</a> BMC Cancer. 29, 313 (2008).</li><li>Pan, X., Yoshida, A., Kawai, A., Kondo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+publicly+available+sarcoma+cell+lines+for+use+in+proteomic+studies.">Current status of publicly available sarcoma cell lines for use in proteomic studies.</a> Expert Rev Proteomics. 13 (2), 227-240 (2016).</li><li>Liverani, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative+approaches+to+establish+and+characterize+primary+cultures:+an+ex+vivo.+3D+system+and+the+zebrafish+model.">Innovative approaches to establish and characterize primary cultures: an ex vivo. 3D system and the zebrafish model.</a> Biol Open. 15 (2), 309 (2017).</li><li>De Vita, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myxofibrosarcoma+primary+cultures:+molecular+and+pharmacological+profile.">Myxofibrosarcoma primary cultures: molecular and pharmacological profile.</a> Ther Adv Med Oncol. 9 (12), 755-767 (2017).</li><li>Blay, J. Y., Ray-Coquard, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sarcoma+in+2016:+Evolving+biological+understanding+and+treatment+of+sarcomas.">Sarcoma in 2016: Evolving biological understanding and treatment of sarcomas.</a> Nat Rev Clin Oncol. 14 (2), 78-80 (2017).</li><li>Dairkee, S. H., Deng, G., Stampfer, M. R., Waldman, F. M., Smith, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+cell+culture+of+primary+breast+carcinoma.">Selective cell culture of primary breast carcinoma.</a> Cancer Res. 55 (12), 2516-2519 (1995).</li><li>Kar, R., Sharma, C., Sen, S., Jain, S. K., Gupta, S. D., Singh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+primary+culture+of+human+ovarian+cancer+cells+to+chemotherapy:+In+vitro+individualized+therapy.">Response of primary culture of human ovarian cancer cells to chemotherapy: In vitro individualized therapy.</a> J Cancer Res Ther. 12 (2), 1050-1055 (2016).</li><li>Ravi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+Tumorigenesis+of+a+Murine+Sarcoma+Cell+Line+in+the+Absence+of+DICER1.">Proliferation and Tumorigenesis of a Murine Sarcoma Cell Line in the Absence of DICER1.</a> Cancer Cell. 21 (6), 848-855 (2012).</li><li>Zhong, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+vegf+gene+knockdown+on+growth+of+the+murine+sarcoma+cell+line+MS-K.">Effect of vegf gene knockdown on growth of the murine sarcoma cell line MS-K.</a> Cells. 16 (6), 625-638 (2011).</li><li>Kidani, T., Yasuda, R., Miyawaki, J., Oshima, Y., Miura, H., Masuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphenol+A+Inhibits+Cell+Proliferation+and+Reduces+the+Motile+Potential+of+Murine+LM8+Osteosarcoma+Cells.">Bisphenol A Inhibits Cell Proliferation and Reduces the Motile Potential of Murine LM8 Osteosarcoma Cells.</a> Anticancer Res. 37 (4), 1711-1722 (2017).</li><li>Sugiyasu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radio-sensitization+of+the+murine+osteosarcoma+cell+line+LM8+with+parthenolide,+a+naturalinhibitor+of+NF-&#954;B.">Radio-sensitization of the murine osteosarcoma cell line LM8 with parthenolide, a naturalinhibitor of NF-&#954;B.</a> Oncol Lett. 2 (3), 407-412 (2011).</li><li>Daigeler, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+in+vitro+effects+of+doxorubicin+on+gene+expression+in+primary+human+liposarcoma+cultures.">Heterogeneous in vitro effects of doxorubicin on gene expression in primary human liposarcoma cultures.</a> BMC Cancer. 29 (8), 313 (2008).</li></ol>

The authors have no conflicts of interest to disclose.

Well defined preclinical models are needed to elucidate the molecular background of tumors, predict poor prognosis, and develop new therapeutic strategies for cancer patients. This is especially important for rare tumors such as STS whose high heterogeneity in terms of morphology, aggressive potential, and clinical behavior challenges our understanding of STS biology and patient management21. Moreover, the few commercial sarcoma cell lines available limits preclinical investigations into this grou...

Soft tissue sarcomas (STS) include a spectrum of heterogeneous lesions of mesenchymal origin accounting for 1% of all solid tumors1,2, and the new World Health Organization classification recognizes the presence of more than 50 different subtypes3. Among these, the most common histotypes are adipocyte sarcoma or liposarcoma and leiomyosarcoma, accounting for 15% and 11% of all adult STS, respectively4,5. Although STS can develop in different parts of the body, the extremities and retroperitoneum are the most common sites, occurring in 60% and 20% of cases, respectively6.
The cornerstone of therapy for localized disease is wide surgical resection, whereas the benefits of adjuvant and neoadjuvant chemotherapy are still unclear and are only considered in selected cases7. In the metastatic setting, chemotherapy is the standard of care but shows limited results8. A better understanding of the molecular biology of STS would help to improve the current differential diagnosis, optimize the available treatments, and identify new targets for therapy.
Within this context, research into cancer has been performed for many years using immortalized cell lines9. These experimental models are normally cultured in standard monolayer supports or implanted into immunodeficient rodents to establish in vivo xenograft models10. Immortalized cell lines represent a manageable and easy-to-store material with which an ample number of research experiments can be performed. They are, in fact, the least expensive and most widely used tool in preclinical studies11.
However, cell-line based cancer models also have some limitations, e.g. prolonged culture in a monolayer system together with an increasing number of passages induces phenotypic and genetic drift, making cells less likely to recapitulate key tumor features. Furthermore, cell line cultures fail to reproduce the cell-to-cell interaction and signaling molecule crosstalk that mimic the biological behavior of tumors and characterize the tumor microenvironment. These issues form the basis of the gap existing between preclinical and clinical results12.
Given the above, the interest in patient-derived primary cultures has increased13. Indeed, the excision of malignant and normal tissue reduces the risk of losing cancer cell phenotype and heterogeneity, thus offering starting material that is more representative of the tumor microenvironment. Moreover, the use of fresh tumor specimens harvested from patients undergoing surgical resection enables us to investigate single tumors and to compare different lesions from the same part of a patient&#39;s body14. For the above reasons, tissue-derived cell cultures provide a valuable material to study tumor pathophysiology and pharmacology15,16,17, especially in STS in which commercially available sarcoma cell lines are limited18. We thus optimized a method to establish a patient-derived STS primary cell culture starting from surgically excised tumor tissue13,19,20. Our method consists of disaggregating the tumor specimen into small tissue fragments followed by overnight enzymatic tissue dissociation to obtain a single cell suspension. The following day, the enzymatic digestion is stopped by adding fresh culture media and the obtained suspension is filtered to remove tissue fragments, cell-aggregates, and excess matrix material or debris. Finally, a fraction of isolated tumor cells is cytospinned onto glass slides, fixed and stored for downstream analysis aimed at confirming the establishment of STS primary culture by cytomorphological, immunohistochemical and molecular cytogenetic evaluation (e.g. FISH analysis of MDM2 amplification represents the standard differential diagnosis for a well-differentiated liposarcoma and the dedifferentiated liposarcoma)4. The remaining cells are seeded into a standard monolayer culture for gene expression profiling and pharmacological studies. All the experiments are performed using low passage primary cultures to avoid the selection of specific cancer subclones, and analyzed by an experienced sarcoma pathologist. We have thus created a fully human STS ex-vivo model13,19,20.This highly versatile, easy to handle model could represent a useful tool for different types of preclinical research, e.g. to identify new molecular markers for diagnosis and prognosis or to gain a deeper understanding of the activity and mechanisms of action of standard and new drugs used in the management of STS.

<table><tbody><tr><td>DMEM High Glucose</td><td>EuroClone</td><td>ECB7501L</td><td></td></tr><tr><td>Fetal bovine serum</td><td>EuroClone</td><td>ECS0180D</td><td></td></tr><tr><td>Glutamine</td><td>Gibco</td><td>25030-024</td><td></td></tr><tr><td>Paraformaldehyde 4% aqueous solution, EM grade</td><td>Electron Microscopy Sciences</td><td>157-4-100</td><td></td></tr><tr><td>Penicillin streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Triazol reagent</td><td>Ambion Life-Technologies</td><td>15596018</td><td></td></tr><tr><td>Trypsin</td><td>EuroClone</td><td>ECB3052D</td><td></td></tr></tbody></table>

establishment of a primary culture of patient-derived soft tissue sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

We designed a simple method to obtain the establishment of a primary culture of patient-derived soft tissue sarcoma and report here an example of results obtained on one specific STS histotype13. The protocol was used for the establishment of primary cultures of different STS histotypes including well-differentiated liposarcoma, dedifferentiated liposarcoma, myxoid liposarcoma, pleomorphic liposarcoma, myxofibrosarcoma, undifferentiated pleomorphic sarcoma, GIST, a...

Watch this Scientific Journal Video about Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma at JoVE.com

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

establishment-primary-culture-patient-derived-soft-tissue

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

14.1K Views.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Video: Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains poor when metastasis has occurred. The Cancer Stem Cell (CSC) theory holds that there is a subset of tumor cells within the tumor that have stem-like characteristics, including the capacity to maintain the tumor and to resist multidrug chemotherapy. Therefore, a better understanding of OS biology and pathogenesis is needed in order to advance the development of targeted therapies to eradicate this particular subset and to reduce morbidity and mortality among patients. Isolating CSCs, establishing cell cultures of CSCs, and studying their biology are important steps to improving our understanding of OS biology and pathogenesis. The establishment of human-derived OS-CSCs from biopsies of OS has been made possible using several methods, including the capacity to create 3-dimensional stem cell cultures under nonadherent conditions. Under these conditions, CSCs are able to create spherical floating colonies formed by daughter stem cells; these colonies are termed &#34;cellular spheres&#34;. Here, we describe a method to establish CSC cultures from primary cell cultures of conventional OS obtained from OS biopsies. We clearly describe the several passages required to isolate and characterize CSCs.

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

The current improvements in therapy against osteosarcoma (OS) have prolonged the lives of cancer patients, but the survival rate of five years remains ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Bioengineering

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Medicine

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Establishing the Primary Culture of Dental Pulp Stem Cells

Primary Culture of Dental Pulp Stem Cells

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an extracted tooth. The neural crest-derived ecto-mesenchymal origin of dental pulp stem cells (DPSCs) bestows a high degree of plasticity that owes to its multifaceted benefits in tissue repair and regeneration. There are various practical ways of harvesting, maintaining, and proliferating adult stem cells being investigated for their use in regenerative medicine. In this work, we demonstrate the establishment of a primary mesenchymal stem cell culture from dental tissue by the explant culture method. The isolated cells were spindle-shaped and adhered to the plastic surface of the culture plate. The phenotypic characterization of these stem cells showed positive expression of the international society of cell therapy (ISCT)-recommended cell surface markers for MSC, such as CD90, CD73, and CD105. Further, negligible expression of hematopoietic (CD45) and endothelial markers (CD34), and less than 2% expression of HLA-DR markers, confirmed the homogeneity and purity of the DPSC cultures. We further illustrated their multipotency based on differentiation to adipogenic, osteogenic, and chondrogenic lineages. We also induced these cells to differentiate into hepatic-like and neuronal-like cells by adding corresponding stimulation media. This optimized protocol will aid in the cultivation of a highly expandable population of mesenchymal stem cells to be utilized in the laboratory or for preclinical studies. Similar protocols can be incorporated into clinical setups for practicing DPSC-based treatments.

Author Spotlight: Advancing Tissue Regeneration and Disease Modeling with Dental Pulp Stem Cells

This article provides a stepwise guide to establish a primary culture of dental pulps stem cells using the explant culture method and characterization of these cells based on ICSCRT guidelines. The cells isolated by this protocol can be considered as mesenchymal stem cells for further applications.

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an ...