Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Summary

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133⁺ putative melanoma stem cells are sorted from the CD133^- bulk using Magnetic Activated Cell Sorting (MACS).

Abstract

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.

Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.

Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133⁺ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133⁺ CSCs and CD133^- bulk, which can be cultivated and functionally analyzed thereafter.

Introduction

Cutaneous malignant melanomas are the least common, yet most deadly type of skin cancer. Due to an increasing incidence, a high grade of malignancy and a rapid dissemination, melanomas now account for 75% of malignant skin tumors related deaths^1,2. Besides surgical excision of the primary tumor, chemotherapy, radiotherapy, immunotherapy and combined chemo- and immunotherapy of metastasized melanoma are the state-of-the-art strategies for melanoma treatment^3,4. However, malignant melanoma is characterized by a poor response to chemotherapeutics (5-12%)^5,6, and only those 50-70% of melanoma patients carrying the BRAF V600E gene mu....

Protocol

The overall scheme of the experiment including the preparation of primary single-cells from tumor tissue, characterization of the primary cell culture, fibroblast depletion and magnetic cell sorting of CD133⁺ and CD133^- melanoma cells is shown in Figure 1.

1. Sample Acquisition

1. Check for ethical approval before considering the next steps.
2. Prepare a 50 ml tube with sterile PBS supplemented with 1% Penicillin-Streptomycin final concent.......

Discussion

In order to remove erythrocyte contaminations from the tumor cell pellet we highly recommend using the red blood cell lysis solution from Miltenyi. The advantages of lysing the erythrocytes over the traditional Ficoll density gradient centrifugation are that it is faster and simpler. Furthermore, contaminations with Ficoll or red blood cells and the loss of tumor cells are avoided. When you observe the growth of fibroblasts in the primary cell culture they should be removed immediately since they normally overgrow the tu.......

Materials

Name	Company	Catalog Number	Comments
			Reagent
Accutase	PAA Laboratories GmbH	L11-007
Amphotericin B solution 250 μg/mL in deionized water	Sigma-Aldrich, Inc.	A2942-50ml
anti-fibroblasts microbeads	Miltenyi Biotec GmbH	130-050-601
CD133/1 (W6B3C1)	Miltenyi Biotec GmbH	130-092-395
Collagenase IV	Sigma-Aldrich, Inc.	C5138
DNase I	Applichem GmbH	A3778.0050
D-PBS without Ca, Mg	PAA Laboratories GmbH	H15-002
Ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA)	Sigma-Aldrich, Inc.	E-7889
FBS Superior	Biochrom	S0615
Indirect CD133 Micro-Bead Kit human	Miltenyi Biotec GmbH	130-091-895	Includes: CD133/1(AC133)-Biotin, anti-Biotin MicroBeads and FcR Blocking Reagent
Penicillin-Streptomycin, liquid (100x)	Invitrogen GmbH	15140-122
Quantum 263	PAA Laboratories GmbH	U15-815
Red Blood Cell Lysis Solution (10x)	Miltenyi Biotec GmbH	130-094-183
			Equipment
Cell strainer (70 μm)	BD Biosciences	352350
gentleMACS C Tubes	Miltenyi Biotec GmbH	130-093-237
gentleMACS dissociator	Miltenyi Biotec GmbH	130-093-235
MACS Separator Multi Stand	Miltenyi Biotec GmbH	130-042-303
MACS Seperation Columns 25 LS Columns	Miltenyi Biotec GmbH	130-042-401
MACSmix Tube Rotator	Miltenyi Biotec GmbH	130-090-753
Natriumchloride	Merck KGaA	567440-1KG
Pre-Separation Filters	Miltenyi Biotec GmbH	130-041-407
QuadroMACS Separator	Miltenyi Biotec GmbH	130-090-976
Rotilabo-syringe filters (0.22 μm, PES)	Carl Roth GmbH & Co. KG	P668.1
Steritop-GP Filter Unit 500 ml	Miltenyi Biotec GmbH	SCGPS05RE