In previous years, three-dimensional (3D) cell culture technology has become a focus of research in tumor cell biology, using a variety of methods and materials to mimic the
As one of the basic techniques utilized to study tumor cell biology, the continual development of tumor cell culture techniques is vital. Traditional cell culture methods use a two-dimensional (2D) monolayer. With continuous improvements being made, this method has become a standard technology in life sciences at present. However, due to the inherent flaws of traditional 2D culture, it fails to correctly imitate the architecture and microenvironments of
As the intersection between tumor cell biology and tissue engineering, 3D
Thus, in the present review, the current preclinical models in cancer research are briefly described in order to promote understanding of the necessity of novel cell culture system. In addition to this, the advantages and challenges of 3D
At present,
Animal models are an important tool for tumor research. Animal model testing is primarily conducted to monitor drug bioavailability, therapeutic efficacy and dose-limiting toxicity (
To resolve these issues, 3D tumor cell culture methods have been developed where the culture environment takes into account the spatial organization and ECM of the cell. The common goal for these methods is to restructure a biomimetic 3D multicellular tumor model, which may bridge the gap between the conventional 2D
MCTS are aggregates of cancer cells grown in suspension or embedded in gels using 3D culture methods. This model partly recapitulates
The suspension culture method was invented to isolate and culture neural stem cells from rat striatal cells in 1970s (
The hanging drop method is a special type of suspension culture (
Device-assisted suspension culture is an improvement of the static suspension culture, depending on several biological devices, including magnetic levitation, spinner or rotational bioreactors and microfluidic devices. The main feature of these bioreactors is to prevent tumor cell adherence or to suspend movement, so that they may grow into MCTS. Additionally, microcarriers or microcapsules are often used in combination to increase the efficiency of cell growth and enhance the protection of the moving cells (
The magnetic levitation method is a novel suspension culture technology invented by n3D Bioscience (
The spinner bioreactor culture method was derived from the study of tumor cells in terms of radiotherapy resistance by Durand and Sutherland (
Microfluidic devices, which allow spatial control over fluids in micrometer-sized channels, have become a valuable tool to further increase the physiological relevance of 3D tumor models (
MCTS in suspension culture lose their complicated ECM microenvironments. The solid tumor cells
Collagen is a widely used material in gel embedding culture (
Alginate is a natural polymer derived from brown seaweed. It gelates in the presence of calcium ions, and is often used for the encapsulation of various types of cells. The main advantage of alginate gel culture is that gelation may be accomplished at room temperature following addition of the cells to the polymer. It allows the cells to mix into the gel-liquid uniformly and grow nondestructively through the process of gelation. The hepatocytes in alginate gel may grow and maintain the function of albumin synthesis (
Matrigel derives from Engelbreth-Holm-Swarm mouse tumor cell-derived basement membrane proteins which include collagen IV, laminin, entactin, perlecan, multiple cytokines and growth factors (
To date, novel gel techniques emerge constantly. Other new commercial gel materials include PathClear Grade Basement Membrane gel (AMS Biotechnology, Ltd., Abingdon, UK), ECM gel (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), ECL Cell Attachment Matrix (Merck KGaA) and Geltrex (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA). In the future, corresponding ECM components may be extracted from different tissues to prepare the gel according to tumor type, in order to improve mimicry of the tumor microenvironments
3D scaffolding is a product of tissue engineering developments. It may act as a surrogate for the missing ECM, representing the available space of tumor tissue, providing the physical support for cell growth, adhesion and proliferation, and causing the cells to form an appropriate spatial distribution and cell-cell or cell-ECM interaction. There are a number of differences between 2D and 3D scaffold culture. A notable comparison is that tumor cells cultured in 3D scaffolds exhibit morphological similarities to tumor cells cultured in human tumor tissues (
In general, the procedures for preparing the scaffolds fall into one of two major categories: 1, natural polymers derived from natural polymer materials, including collagen, chitosan, glycosaminoglycans (mainly hyaluronic acid), fibroin, agarose, alginate, and starch (mainly used as additives); and 2, synthetic polymers, containing polyglycolic acid, polylactic acid, polyorthoester and their copolymers or blends, as well as the aliphatic polyester polycaprolactone (
A number of highly influential studies regarding 3D scaffolding culture in drug research and CSCs have been reported. Dhiman
Different approaches offer different advantages and disadvantages, as summarized in
In summary, the biological influence of the 3D microenvironment on tumor cell differentiation, progression, metastasis and chemotherapy-resistance has gained increased recognition. 3D culture, which ranges from the simple cell spheroid model to complex tissue-engineered constructs, serves an increasingly important function in tumor cell biology research. Gel embedding and scaffolds have a number of advantages in simulating the 3D structure of tumor cells
The present study was supported the Key Health Project of the Nanjing Military Region (grant no. 12MA031).
Methods available for MCTS formation. These methods include (A) static suspension; (B) hanging drop methods; (C) spinner bioreactor; (D) rotational bioreactor; (E) magnetic levitation; (F) microfluidic system; and (G) gel embedding. (H) A classic MCTS was observed by inverted phase contrast microscope (scale bar, 100 µm). MCTS, multicellular tumor spheroids.
Comparison of tumor cells morphology between 2D and 3D scaffold culture. U87 cells in 2D (left) and 3D scaffold (right) in a scanning electron microscope image (scale bars, 100 and 10 µm). 2D, two dimensional; 3D, three dimensional.
Previous 3D cell culture models to simulate the tumor microenvironment and assess drug. Arranged according to tumor type.
Author, year | Model | Cells | 3D model | Results | (Refs.) |
---|---|---|---|---|---|
Talukdar and Kundu, 2012 | Breast cancer | MDA-MB-231 | Non-mulberry silk fibroin protein scaffolds | Notably higher drug concentrations are required to achieve a comparable reduction in cell viability and invasive potential in 3D cultures than 2D cultures | ( |
Chen |
MCF-7 | Collagen scaffolds | High-level expression of CSC-associated properties of MCF-7 cells cultured in 3D were confirmed | ( |
|
Dunne |
MCF-7, BT474, SKBR3 | Scaffolds | Breast cancer cells grown in 3D on the decellularized scaffolds demonstrated reduced sensitivity to doxorubicin relative to 2D cell culture | ( |
|
Maguire |
MCF-10 | Multicellular tumor spheroids | Genetic dependencies can be uncovered when cells are grown in 3D conditions similar to |
( |
|
Sha |
Gastric cancer | BGC-823 | Multicellular tumor spheroids | Anti-EGFR-iRGD may improve anticancer drugs, such as doxorubicin, bevacizumab, nanoparticle permeability and efficacy throughout the tumor mass | ( |
Kundu |
Hepatocarcinoma | HepG2 | Tasar silk fibroin scaffolds | 3D multicellular model demonstrates insight into hepatocarcinoma progression and offers a prediction of cellular response to transfection efficacy, drug treatment and therapeutic intervention | ( |
Xu |
Lung cancer | Primary lung cancer cells, SPCA-1 | Spheroids in device-assisted culture | Developed a high-throughput model for assessing drug sensitivities |
( |
Simon |
A549 | Hydrogels | 3D model can help regulate the exposure of oxygen to subpopulations of cells in a tissue-like construct either prior to or following therapy | ( |
|
Stratmann |
HCC827, A549 | Decellularized scaffolds | Quantitative read-outs for proliferation, apoptosis and invasion were established in the complex 3D tumor model | ( |
|
Lee |
Ovarian cancer | 1847, A2780, CaOV3, COV644, EFO27, ES-2, FUOV1, HEY, IGROV1 | Multicellular tumor spheroids | 3D cell culture is an improved reflection of the histological, biological and molecular features of tumors compared with primary cultures in 2D. In terms of chemosensitivity, 7 out of 11 cell lines were more resistant in 3D models. 7 out of 11 cell lines also demonstrated increased resistance to paclitaxel | ( |
Shin |
HEY | Multicellular tumor spheroids | Taxol-induced apoptosis was detected in monolayer conditions but not in spheroid cultures. | ( |
|
Loessner |
OV-MZ-6 | Multicellular tumor spheroids | Combinatorial approaches of paclitaxel and KLK/MAPK inhibition may be more efficient than chemotherapeutics alone | ( |
|
Yang and Zhao, 2011 | A2780, A2780/DDP, SK-OV-3 | Nanofiber scaffolds | Ovarian cancer cells demonstrated between two and five-fold higher drug resistance (fluorouracil, paclitaxel and curcumin) relative to 2D culture | ( |
|
Fitzgerald |
Prostate cancer | PC3, LNCaP | Collagen-based scaffold | The two cell lines in 3D demonstrated higher resistance to docetaxel. Non-viral delivery vectors, lipofectamine and a modified cyclodextrin, mediated gene knockdown in this 3D model | ( |
Xu |
LNCaP | HA-based gels | The hydrogel serves as a diffusion barrier for nanoparticles. Cells cultured in 3D were more resistant to docetaxel treatment relative to 2D | ( |
|
Lv |
Glioma | Primary cells, U87 | Collagen scaffolds | 3D-cultured cells also demonstrated enhanced resistance to chemotherapeutic alkylating agents, with a much higher proportion of glioma stem cells and upregulation of MGMT | ( |
Ma |
U251 | Polystyrene scaffolds coated with laminin | The results indicate the influence of 3D context on integrin expression, specifically, the upregulation of the laminin-binding integrin's alpha 6 and beta 4 | ( |
|
Pedron |
U87 | HA gels | Clustering of glioma cells was observed exclusively in HA gels and expression of malignant genes was revealed to vary bi-phasically with incorporated HA content. | ( |
|
Munson |
RT2, U87, C6, 9L | HA-collagen gels | Interstitial flow in this 3D model increases glioma invasion by a CXCR4-dependent mechanism | ( |
|
Fong |
Ewing sarcoma | TC-71 | Electrospun poly(e-caprolactone) scaffolds | Ewing sarcoma cells cultured in 3D were more resistant to traditional cytotoxic drugs, and exhibited remarkable differences in the expression pattern of the IGF-1R/mammalian target of rapamycin pathway | ( |
2D, two-dimensional; 3D, three-dimensional; CSC, cancer stem cell; KLK, kallikrein-related peptidases; MAPK, mitogen-activated protein kinases; HA, hyaluronic acid; MGMT, O-6-methyltransferase; CXCR4, C-X-C motif chemokine receptor 4; IGF-IR, insulin-like growth factor receptor.
Comparison of various methods of 3D tumor cell culture.
Method | Advantages | Disadvantages |
---|---|---|
Static suspension culture | Relatively simple, low cost, spheroid production is easily accessible, reproducible | Continuous passage culture difficult, relatively labor intensive, only autocrine ECM |
Hanging drop | Control of spheroid size, low cost if using standard 96-well plate, uniform spheroid size, homogenous spheroids, suitable for high-throughput testing | Expensive if using specialized plates, long-term culture difficult, small culture volume, medium exchange difficult |
Magnetic levitation | Relatively simple tool, efficient, does not require any specialized media | Colors the 3D culture brown and limit certain applications, numerous cells also adhere to the bottom of the plate |
Spinner/rotational based approaches | Mass production, long-time culture, relatively simple | Require specialized equipment, not possible to control uniformity of spheroid (size, composition), exposed to high shear force |
Microfluidic device | Suitable for control of spheroid (size, parameters), continuous perfusion for faster spheroid formation | Difficulty collecting cells for analysis, require expense and complicated equipment |
Gel embedding | Resemblance to the |
Poor mechanical properties, certain components of natural gel are variable and undefined, lacks vasculature |
Scaffolds | Maximum resemblance to the |
Expensive for large-scale production, trouble in cells dissociation from scaffold, complex operation |
3D, three dimensional; ECM, extracellular matrix.