用于检测基因敲减效率和细胞系建立3D培养方法

Jan Strnadel; Sang Myung Woo; Sunkyu Choi; Huawei   Wang; Marian Grendar; Ken Fujimura

doi:10.21769/BioProtoc.2874

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

3D Culture Protocol for Testing Gene Knockdown Efficiency and Cell Line Derivation

用于检测基因敲减效率和细胞系建立3D培养方法

Jan Strnadel email

发布: 2018年06月05日第8卷第11期 DOI: 10.21769/BioProtoc.2874 浏览次数: 9923

评审: Chiara AmbrogioMauro Sbroggio'Enrico Patrucco

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Cancer Research, featuring study using the protocol.

Apr 2017

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

人婴幼儿血管瘤中干细胞、内皮细胞和周细胞的分离

Lan Huang and Joyce Bischoff

2020年01月20日 4045 阅读

用于人结直肠上皮类器官和类肿瘤的低粘度基质悬浮培养

Tao Tan [...] Oliver M. Sieber

2022年04月20日 3130 阅读

来自骨髓增生性肿瘤患者的造血祖细胞的血小板生成素不依赖性巨核细胞分化

Chloe A. L. Thompson-Peach [...] Daniel Thomas

2023年01月20日 1288 阅读

Abstract

Traditional 2D cell cultures with cells grown as monolayers on solid surface still represent the standard method in cancer research for drug testing. Cells grown in 2D cultures, however, lack relevant cell-matrix and cell-cell interactions and ignore the true three-dimensional anatomy of solid tumors. Cells cultured in 2D can also undergo cytoskeletal rearrangements and acquire artificial polarity associated with aberrant gene expression (Edmondson et al., 2014). 3D culture systems that better mimic the in vivo situation have been developed recently. 3D in vitro cancer models (tumorspheres) for studying cancer stem cells have gained increased popularity in the field (Weiswald et al., 2015). Systems that use matrix-embedded or encapsulated spheroids, spheroids cultured in hanging drops, magnetic levitation systems or 3D printing methods are already being widely used in research and for novel drug screening. In this article, we describe a detailed protocol for testing the effect of shRNA-mediated gene silencing on tumorsphere formation and growth. This approach allows researchers to test the impact of gene knockdown on the growth of tumor initiating cells. As verified by our lab, the protocol can be also used for isolation of 3D cancer cell lines directly from tumor tissues.

Keywords: 3D culture (三维培养)

Tumorspheres (肿瘤球)

Cancer cell line (癌细胞系)

shRNA gene silencing (shRNA基因沉默)

Background

3D in vitro cancer cell models represent a bridge experimental method between cell lines and tumors grown in vivo (Pampaloni et al., 2007; Weiswald et al., 2015). 3D characters of solid tumors with heterogeneous access to nutrients or oxygen can only be effectively mimicked by 3D culture systems. In recent years, protocols for tumorsphere culture gained lot of interest. A tumorsphere can be described as a solid, spherical object created from a single progenitor or stem cell. For tumorspheres formation assays, cells are seeded and grown in serum-free media in ultra-low attachment plates (non-adherent conditions), which allows enrichment of cancer cells with stem/progenitor properties (Johnson et al., 2013). Tumorspheres generated from freshly isolated tumor tissue are of special interest in the field because cells from established cell lines typically differ from the primary tumor due to mutations and abnormalities gained during multiple rounds of in vitro passaging. Hereby, we present an optimized protocol for 3D culture-based primary tumor cell isolation and the use of 3D culture to assess the effect of gene silencing on the growth of tumor-initiation cells.

Materials and Reagents

Eppendorf tube
Pipette tips
Vials
Plastic bottles
50 ml Falcon tube
Petri dishes with clear lid (Fisher Scientific, Fisherbrand, catalog number: FB0875712 )
6-well plates, Corning Costar Ultra-Low Attachment (Corning, catalog number: 3471 )
15 ml Falcon tubes (Corning, Falcon^®, catalog number: 352196 )
70 µm cell filter (cell strainer - Corning, Falcon^®, catalog number: 352350 )
Ice
Plastic pipette
24-well plates, Corning Costar Ultra-Low Attachment (Corning, catalog number: 3473 )
6-well plates (Corning, catalog number: 3506 )
Serological pipettes
Surgical razor blades (FisherBrand High Precision # 22 Style Scalpel Blade, Fisher Scientific, Fisherbrand^TM, catalog number: 12-000-161 )
Sterile pipets (10 ml) (FisherBrand Sterile Disposable Standard Serological Pipets, Fisher Scientific, Fisherbrand^TM, catalog number: 13-678-14A )
Cryovials (General Long-Term Storage Cryogenic Tubes 1 ml, Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 5000-1012 )
(optional) Mission pLKO.1-puro-CMV-TurboGFP Positive control Transduction Particles (Sigma-Aldrich, catalog number: SHC003V )
EDTA (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 15575-020 )
PBS pH 7.4 (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010023 )
Trypsin EDTA (0.05%) (Thermo Fisher Scientific, Gibco^TM, catalog number: 25300054 )
SureEntry transduction reagent (QIAGEN, catalog number: 336921 )
Clorox Bleach (Veritiv, catalog number: 30966 )
Heparin Sodium Salt (Sigma-Aldrich, catalog number: H3149-10KU )
DMEM/F12, Glutamax (Thermo Fisher Scientific, Gibco^TM, catalog number: 10565018 )
Penicillin/Streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15070063 )
Fetal bovine serum (Thermo Fisher Scientific, Gibco^TM, catalog number: 26140079 )
B27 supplement 50x (serum free) (Thermo Fisher Scientific, Gibco^TM, catalog number: 17504044 )
bFGF – human Fibroblasts Growth Factor 147 basic (animal free) (Gemini Bio-Products, catalog number: 300-805P )
EGF– Epidermal Growth Factor (human) (Gemini Bio-Products, catalog number: 300-110P )
ROCK inhibitor (Y-27632) (STEMCELL Technologies, catalog number: 72304 )
Heparin solution (STEMCELL Technologies, catalog number: 07980 )
BD Matrigel Matrix Growth Factor Reduced (BD Biosciences, catalog number: 356230 ) or Cultrex^® 3-D Culture Matrix^TM Reduced Growth Factor Basement Membrane Extract (Trevigen, catalog number: 3445-001-01 )
Notes:
1. Aliquot Matrigel or Cultrex Matrix into single-use aliquots in a sterile hood into sterile Eppendorf tubes. We recommend 0.5 ml of Matrigel per Eppendorf tube. When combined with 1.5 ml of ice-cold media, this amount is sufficient for 3D culture using one well in a six-well plate.
2. Use chilled pipettes and have Eppendorf tubes on ice during procedure.
3. Store at -80 °C. Thaw on ice or in the refrigerator overnight before use.
Hibernation medium (Hibernate^TM-A, Thermo Fisher Scientific, Gibco^TM, catalog number: A1247501 )
Collagenase IV (Sigma-Aldrich, catalog number: C8051-100MG or similar)
Trypan Blue (TC-10, Bio-Rad Laboratories, catalog number: 1450013 )
Antibiotic-Antimycotic (100x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15240096 )
Dispase (1 U/ml, STEMCELL Technologies, catalog number: 07923 )
EDTA (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 15575020 )
Accutase (STEMCELL Technologies, catalog number: 07920 )
DMSO (Sigma-Aldrich, catalog number: D2438-50ML )
Ethanol (70% solution, Fisher Scientific, Fisher BioReagents^TM, catalog number: BP8201500 )
Culture medium (see Recipes)
CSC medium (see Recipes)

Equipment

Centrifuge (temperature controlled, VWR^® benchtop general purpose centrifuge) (VWR, catalog number: 10830-764 )
Pipettes
Forceps
CO₂ Cell incubator (BINDER, model: CB 160 )
Biological Safety Level (BSL-2) laminar flow hood (Esco, EscoTechnologies, USA)
TC20 Automated cell counter (Bio-Rad)
Mr. Frosty Freezing Container (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 5100-0001 )
Forma^TM 7000 series Ultra Low Temperature Freezers (Thermo Fisher Scientific, Thermo Scientific^TM, model: TFM#902 Series )
Liquid nitrogen storage tank
Nikon TE-2000E D-Eclipse Csi confocal microscope running Nikon Elements software (Nikon, model: Eclipse TE2000-E )

Software

Nikon microscope operating software
ImageJ or similar image processing software (https://imagej.nih.gov/ij/)
GraphPad Prism 6.0 software from GraphPad Software, USA
(https://www.graphpad.com/scientific-software/prism/)
R software (R Core Team, 2015 R: A language and environment for statistical computing)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Strnadel, J., Woo, S. M., Choi, S., Wang, H., Grendar, M. and Fujimura, K. (2018). 3D Culture Protocol for Testing Gene Knockdown Efficiency and Cell Line Derivation. Bio-protocol 8(11): e2874. DOI: 10.21769/BioProtoc.2874.