Journal of Cytology & Tissue Biology Category: Clinical Type: Research Article
3D Cell Culture on VitroGel System
*Corresponding Author:John Huang
Department Of Biotechnology, TheWell Bioscience, New Jersey, United States
Received Date: May 29, 2019 Accepted Date: Jun 24, 2019 Published Date: Jul 01, 2019
3D cell culture; Cancer; Drug screen; Hydrogel; VitroGel
The advent of three-dimensional cell culture began in the 1980s after researchers began to understand the various physiological differences that occur when cells are cultured ex vivo. One of the earliest 3D scaffolds was developed in 1988 by Eric Simon, who was conducting an NIH sponsored feasibility study for fibrous scaffold inserts to facilitate 3D tissue culture . Although he concluded that the electrospun polycarbonate scaffolds were inferior to standard 2D tissue culture, this initial study identified several of improvement including lack of mechanical strength, low-level adhesion of the cells to the scaffold that resulted in unintentional dissociation, and inability to image cells within the scaffold. Since that time, researchers have developed various scaffolds that have successfully addressed these issues and promoted cell growth. For example, although the initial polymer scaffold developed by Simon was unsuccessful, scientists have since found other ways to develop polymer scaffolds that very closely mimic the extracellular matrix and allow cells to settle in and incorporate into pre-fabricated geometric scaffolds with consistent pore sizes . Some of the more common materials to make these scaffolds include polystyrene and polycaprolactone, which have been popular for use in bone regeneration studies because to generate scaffolds that closely mimic the structure of the bone matrix . There are also scaffolds known as biological scaffolds which are built using components that are found naturally in the in vivoextracellular matrix. For example, collagen matrices, which are primarily intended to mimic cartilage for the purposes of joint regeneration [6,7], can be altered for use in other applications such as ligament or skin regeneration. However, they have limitations related to their rigid mechanical properties [8,9].
Cells cultured in 3D environments have many advantages over 2D culture in terms of mimicking the in vivo environment. They exhibit more realistic distribution and cellular access to oxygen and nutrients/media components, they experience increased cell-to-cell and cell-to-ECM interactions , and they can develop district zones of different cell proliferation or differentiation ability . However, research has demonstrated that a single cell line cultured in different 3D systems can exhibit different cellular behavior [12-14]. Conversely, within a single 3D system, some cells may have a positive response, while others may fail to thrive, making it different to study multicellular tissues within a single 3D model . Moreover, many 3D scaffolds cannot be used in both in vitro and in vivo settings, making it difficult to conduct crossover studies or to culture cells in vitro for the purposes of transplantation .
One type of scaffold that may be able to overcome some of the limitations of previous scaffolds is hydrogel. A hydrogel is a versatile macromolecular polymer gel comprised of crosslinked polymer chains synthesized from hydrophilic monomers. Hydrogels can be used to generate multi-layer tissue-like structures during cell culture: different cell types can be embedded into separate hydrogel suspensions and layered on top of each other. Permeable supports can also be incorporated to simulate various air-liquid interfaces in an in vitro manner . The hydrogel system can be composited with growth factors and biological functional ligands, which is able to better promote cellular function, leading to increased viability and recapitulating a more accurate in vivo like cellular environment. Despite these many advantages, hydrogels also have several limitations. In research, hydrogels are often too opaque to allow for live imaging, instead requiring dissociation of cells for successful imaging [16,18]. With regard to clinical applications, hydrogels tend to have low tensile strength which can limit their use in load-bearing applications like bone engineering. The tensile strength can also impede drug discovery applications; it can cause premature dissolution of the hydrogel . Moreover, hydrogels have not been ideal in applications involving drug delivery, particularly if the drugs are hydrophobic. Their water content and large pore size can impede drugs that need to be released over longer periods of time, releasing the drug too quickly. Each of these issues significantly restricts the practical use of hydrogel-based drug delivery therapies in the clinic . Additionally, most current hydrogel systems are limited by such conditions as poor scaffold structure, unknown growth factors, and undesirable pH or temperature of the pre-gel solution, which lead to the challenges of complex operation, difficulties in downstream analysis, and product reproducibility.
VitroGel is a ready-to-use, xeno-free tunable hydrogel system which closely mimics the natural extracellular matrix environment. The hydrogel system is room stable at room temperature, has a neutral pH and is transparent, permeable and compatible with different imaging systems. The solution transforms into a tunable hydrogel matrix simply by mixing with cell culture medium, which shorten the operation time from hours to 20 min Cells cultured in this system can also be easily harvested for downstream analysis. The hydrogel can also be tuned to be injectable for in vivo studies. From 2D coating and 3D culture to animal injection, VitroGel makes it possible to bridge the in vitro and in vivo studies with the same platform system. VitroGel offers solutions to many of the existing problems presented by hydrogels. Mechanical properties and rate of hydrogel can be adjusted and the biological functional of the hydrogel system can be modified to mimic the endogenous physiological microenvironment. Since different cell types prefer different tissue-specific microenvironments, the hydrogel conditions can be optimized for different cell types and culture media, allowing for the ideal environment to be created for each cell type in multicellular applications. In addition to offering these solutions, VitroGel can be used in 2D applications and 3D in vitro and in vivo applications, offering a single platform by which to bridge the gap between traditional 2D system and in vivo study.
MATERIALS AND METHODS
2D coating culture method
3D culture method
PANC-1 and HCT116 cells were cultured in both VitroGel 3D (TWG001, TheWell Bioscience, NJ) and VitroGel 3D-RGD (TWG002, TheWell Bioscience, NJ), while OP9 and U-87 cells were cultured in both VitroGel 3D-RGD, and the newer VitroGel RGD-PLUS (TWG003, TheWell Bioscience, NJ).
Since OP9 cells have a GFP reporter, we conducted live cell imaging. The images were taken using a Leica TCS SP8 scanning confocal microscope with z-stack function at 10X and 20X magnification and processed by LAS X software and ImageJ.
RESULTS AND DISCUSSION
VitroGel 3D can mimic the tumor microenvironment
PANC-1 cells were cultured in dilutions of hydrogel solution. At day 2 (Figure 1A) in the 3D cell culture, cells were able to distribute homogeneously within the hydrogel (Figure 1), showing no difference between dilutions (not shown). Cells maintained through day 7 showed significant concentration-based differences were seen based on the concentration of the hydrogel (Figures 1B-1D). Individual cells formed colonies in each condition, however, the 1:1 and 1:3 dilutions of hydrogel (Figures 1C,1D) allowed for more rapid division and expansion of the single cells, developing larger 3D colonies. Another critical component of 3D culture comes from the development of an in vivo like extracellular matrix which can be recapitulated in vitro. Act in and nuclear staining (Figures 2A-2C) reveal increasing complexity and dispersion of the ECM generated by the cell-cell contact of PANC-1 cells when maintained in VitroGel 3D-RGD hydrogel. Although different cells may exhibit a distinct response, our studies demonstrated that PANC-1 cells grow into larger spheres with higher dilutions of the hydrogel (Figures 2B,2C). A concern of growing 3D spheres comes from the notion that the center of the sphere may become necrotic . To verify that cells are viable in our 3D system, we conducted a live/dead assay. This assessment revealed no significant death in each of the three hydrogel dilutions (Figures 2D-2F), indicating that the growth rate and complexity of PANC-1 3D spheres cultured in VitroGel more accurately mimic the in vivo environment, thus allowing for better studies for pancreatic cancer.
Treatment for glioblastoma has also been difficult to develop because two-dimensional culture systems fail to mimic the tumor microenvironment. Tumor progression depends critically upon the interactions between the tumor cells and their microenvironment . The tumor microenvironment is heterogeneous and dynamic; it consists of extracellular matrix, stromal cells, immune cells, progenitor cells, and blood and lymphatic vessels, which is impossible to recreate in a 2D environment. Various studies have shown that tumor cells cultured two-dimensional environments exhibit distinct differences in cell proliferation ability, resistance to apoptosis, differentiation ability, invasive ability and even biomarker expression [24,25]. Moreover, it isn’t enough to simply study tumor-derived cells; tumors are inherently three-dimensional, and that morphology plays a role in tumor progression, namely angiogenesis, a critical factor in malignancy . Additionally, cancer cells with properties similar to stem cells have been found in glioblastoma and are thought to contribute the high resistance to conventional treatments, and the high recurrence rate . Therefore, accurate and effective development of treatments for glioblastoma will require 3D models that closely mimic the cell-cell interactions exhibited in 3D tumors.
U-87 cells are a line of human primary glioblastoma cells  that are commonly used in studies aimed at understanding causality and progression of glioblastoma. To investigate whether our 2D VitroGel system is conducive to the growth of U-87 cells in a two-dimensional system, we cultured them in various dilutions of two of our VitroGel products. U-87 cells were harvested from a human glioblastoma tumor, and although it is in their inherent nature to cluster, when they are cultured in standard 2D culture, they adopt fibroblast-like morphology and only make small aggregates (Figure 3A). In the VitroGel hydrogels, the cells exhibited substantially more spheroid clustering, even at the lower dilutions (Figures 3B-3E). In the 1:3 dilutions of both hydrogels (Figures 3C,3E), the colonies formed are smaller, but in the new softer VitroGel RGD-PLUS, the cell clusters exhibit an interesting morphological characteristic: they form bridges between the clusters (Figure 3E). Culture of U-87 cells on 2D VitroGel demonstrates that this system promotes spheroid morphology even in a 2D environment.
A tunable hydrogel suitable for drug screening applications
We evaluated the viability and survival of HCT116 cells in the VitroGel system for 2D coating, 3D culture, and drug testing. Both VitroGel 3D and VitroGel 3D-RGD (the RGD peptide modified hydrogel system for better cell adhesion) were used in our studies. HCT116 cells are a line of human colorectal carcinoma cells commonly used in colon cancer studies and therapeutic drug development. These studies established the VitroGel system as a viable means by which to study drug development using HCT116 cells as a model for colorectal cancer.
We evaluated HCT116 cells using both versions of the VitroGel system (Figure 4A): the standard VitroGel-3D system, which is ideal for suspension cells, and the RGD-modified version, which is contains the RGD peptide modification, a peptide present in all major extracellular matrix proteins, and which can promote cell adhesion to biomaterials such as Hydrogels . Cells were evaluated 24 hours after seeding cells and again 7 days later. When using the 2D coating method (Figures 4B,4C,4E,4F), in which the VitroGel hydrogel is allowed to set before cells are seeded, cells adopted a different morphology depending on which version of the VitroGel system was used. In the VitroGel 3D setup (Figures 4A,4D), cells acquired a spheroid shape, albeit small, after 24 hours, and continued to increase in size out to 7 days. Like the standard VitroGel 3D, cells on VitroGel 3D-RGD also began to form spheroid cell clusters after 24 hours. However, unlike the standard hydrogel, cells spread out after 7 days, adopting morphology reminiscent of the 2D culture on standard treated culture plastic. When using the 3D cell culture method in the VitroGel 3D-RGD system, in which cells are seeded into the hydrogel prior to the setting of the gel, HCT116 cells are found to distribute homogenously within the hydrogel. Strength of the hydrogel can affect its mechanical properties, and thus cell viability and response within the hydrogel. We analyzed growth potential (Figure 5) and viability (Figure 6) of HCT116 cells in three different hydrogel strengths (concentrations): 1:0 dilution (mostly hydrogel), and 1:1 and 1:2 dilutions of hydrogel. After 7 days, the 1:1 hydrogel dilution allowed for big colonies (Figure 5B), while other dilutions only allowed for small cell clusters/colonies with abnormal morphology (Figures 5A,5C). Analysis of cell viability by live/dead assay showed that there was no significant cell death in any of the hydrogel dilutions (Figures 6A-6C), despite reduced colony size. Although the cells in the 1:0 and 1:2 dilutions (Figures 6B,6C) aren't exhibiting many dead markers, their morphology indicates they may be dying if not already dead. In the 1:1 dilution, there are more cells, and although they have a flatter morphology commonly associated with poor cell health, it is thought that the increased cell-matrix interaction allowed in scaffolds prevents the spheroid morphology seen in ultra-low adhesion plates used in 2D culture. This morphology might indicate a good cell-matrix interaction which could be good to mimic in vivo situation.
5-Fluorouracil (5-FU) is a common anti-cancer drug used to treat several cancers including colorectal cancer. While it has some demonstrated some success in colon cancer, more recent studies have pointed out that many patients develop a resistance to 5-FU , and other studies have found that 5-FU only acts on a small subset of colon cancers that have a mismatch repair gene mutation . Testing 5-FU on HCT116 cells using traditional 2D cell culture system juxtaposed against the 3D culture system demonstrated that the effect of the 5-FU is insignificant (Figure 7). We found that in 2D cultures, drug efficiency increased as the drug concentration increased from 10µM to 50µM (Figures 7A-7D). However, in the 3D cell culture system, there was no significant drug effect at any stage (Figures 7E-7H). Only in the case of 50µM (Figure 7H) is there any noticeable effect, reduced size of cell colonies clearly observable. Quantification of cell death with a live/dead assay (Figure 7I) showed increasing levels of dead cells in the 2D cultures, suggesting an effectiveness of 5-FU in killing cancer cells. However, in 3D culture, there was not a significant level of dead cells at any stage; nearly non-existent until 50µM. Quantification of live cells using ImageJ showed a steady decline with increased drug concentration. However, in 3D cultures, this decline was not present, demonstrating the importance of 3D culture for accurate interpretation of drug effect.
In 3D cell culture, we studied the cell morphology, growth, and survival in response to different hydrogel strength (hydrogel dilution). The results of live/dead assay showed that there was not an increase in cell death, suggesting that the reduction in total number of cells in the lower and higher hydrogel dilutions resulted from the mechanical interactions of the cells with the hydrogel. Previous studies have found that the number of live cells is dependent upon substrate stiffness, while the number of dead cells is unrelated, suggesting that the stiffness of the hydrogel can affect proliferation [11,35]. In fact, studies conducted on hepatocellular carcinoma cells demonstrated that the mechanical properties of a matrix can not only promote proliferation, but also can moderate chemotherapeutic response .
In figure 5, we observed varying levels of Actin in different parts of the clusters and even between clusters in different 3D scaffold stiffness. Some clusters have more Actin than others, and Actin localization in scaffolds of different stiffness varies. Although we used Actin simply to observe cluster morphology (i.e., flat vs. spherical), it should be noted that there are studies that show that actin levels can fluctuate in cancer cells at different stages [36-38]. In research, Actin is useful as a morphology marker because it is a homeostatic gene, but its actual function relates to multiple cellular processes from contraction to cell division and motility. One study found that actin filaments located at the leading edge of cancer cells are motile in nature and have a higher rate of turnover . Another study found that actin plays a major role in both pre-malignant and malignant breast tumor cells by first controlling cell stiffness to promote proliferation and then cell softness to allow for invasion . The variation in Actin localization and expression levels between different scaffold rigidities concomitant with studies that show that Actin expression varies in cancer cells suggests that the 3D scaffold can affect the motility of the cell. Because cells respond uniquely to matrix rigidity, it is likely that each cell line will have to be optimized prior to experimentation. However, the wide array of flexibility this system offers with regard to culture medium compatibility and its potential to be used in vitro and in vivo make VitroGel a great option for drug testing, especially for tumor applications.
Three-dimensional hydrogels are more suited to promoted intercellular networking
However, for all of their therapeutic potential, they have one major setback: in vitro, their proliferation rate tends to slow drastically, and they begin to senesce readily, making them difficult to expand enough for therapeutic use . Some studies have suggested that adjusting the glucose concentration in the culture media can mitigate some of the senescence , but this means culturing the cells in an environment that is not clinically relevant and which could ultimately alter their genetic stability. Studies have been conducted on the effect of 2D cell culture on the expression patterns and they have found that 2D culture tends to result in the overall flattening of the cytoskeleton and nucleus which in turn results in a change in gene and gene expression and rearrangement of the extracellular matrix. Three-dimensional cultures have proven to be much more successful in maintain the ECM microenvironment and produce more uniform, reproducible results and have demonstrated more success with clinical applications including transplantation.
OP9 cells are a characterized line of murine bone marrow stromal cells that do not produce functional macrophage colony-stimulating factor (M-CSF) due to an osteopetrotic mutation in the gene encoding M-CSF . OP9 cells were initially used to coculture mouse embryonic stem cells to induce their differentiation into blood cells, but they have been characterized as mesenchymal stem cells . Here we discuss culturing of OP9 mesenchymal stem cells in the VitroGel tunable hydrogel system including the new VitroGel RGD-PLUS. OP9 cells in VitroGel 2D culture exhibit cell clustering and healthy cell morphology. We evaluated OP9 cells in 2D culture in both the original VitroGel 3D-RGD and the new VitroGel RGD-PLUS. In standard 2D culture, OP9 cells, like many mesenchymal cell types, cells adopt a fibroblast like shape and fail to cluster (Figure 8A). Mesenchymal stem cells in this environment don’t proliferate well and can lead to senescence. Moreover, they exhibit the characteristic bright spotting in the cell culture commonly associated with stages of apoptosis. To understand optimal conditions for OP9 culture within our tunable system, we cultured these cells various dilutions of VitroGel. In the VitroGel 3D-RGD (Figures 8B-8D), cells form clusters in all dilutions after just 2 days of cell culture. With the newer VitroGel-3D-PLUS (Figures 8E-8G), cells are even more robust, forming large colonies and exhibiting a healthy MSC morphology at the lowest dilution. A major factor of the in vivo 3D microenvironment is the ability of cells to form an intercellular network. This characteristic is typically not possible in two-dimensional culture and can vary with three-dimensional culture. To understand whether our hydrogel system can facilitate this, we cultured OP9 cells in three-dimensional cultures with two of our hydrogels. With VitroGel 3D-RGD, after 7 days of culture, cells have settled into the 3D hydrogel (Figures 9A-9E,9G), but cells cultured in VitroGel RGD-PLUS have already begun to make intercellular connections (Figures 9F,9H).
Figure 9: Three-dimensional culture of OP9-GFP cells in both VitroGel 3D-RGD and VitroGel RGD-PLUS. While VitroGel 3D-RGD promotes cell expansion (A-D), the new VitroGel RGD-PLUS has better support for OP9 cell proliferation and cell-cell communication. Stronger cell-matrix interactions help the cells to form the cell-networking structures (E-H). (images A,B,C,D,G,H at 20X; images E&F at 10X).
Our studies indicate that our new VitroGel 3D RGD-PLUS can be an ideal three-dimensional scaffold for cellular applications that rely on intercellular networks. Cellular differentiation and identity are regulated by complex cell-cell communication which can only be achieved at a short range by diffusible ligands; a behavior which can be difficult to model in vitro. It can lead to variability between in vitro and in vivostudies which can hinder translational studies . For tissue regeneration, especially mesenchymal differentiation, the ability to communicate with surrounding cells is absolutely necessary. Cell communication commonly happens at relatively short-range via diffusible factors between cells, facilitated by the close contact provided by a three-dimensional environment. However, longer range signals require a cell communications network. Multiple studies have demonstrated that different kinds of scaffolds and different elasticities can drive differentiation down different pathways. VitroGel 3D provides a platform that is adjustable enough to fit any of the applications, reducing variability and streamlining the process.
To further investigate the ability of VitroGel RGD-PLUS in promoting inter cellular networking, we culture U-87 MG glioblastoma cells in three-dimensional culture at different dilutions. We found that in both concentrations of the original VitroGel 3D-RGD, cells settle into the hydrogel after only two days (Figures 10A-10C), but after seven days (Figures 10D,10E), their overall spread has not changed, and they do not exhibit any intercellular network connections (Figure 10F,10G). In the VitroGel RGD-PLUS, not only do cells settle into the hydrogel by day two, but they also exhibit markedly different morphology from the cells in the VitroGel 3D-RGD: they are more elongated. After 7 days, they exhibit broad cell-cell connections within the 3D hydrogel (Figures 10H,10I) in clusters much larger than the VitroGel 3D-RGD (Figure 10J).
Figure 10: U-87 MG cells cultured in both VitroGel 3D-RGD and VitroGel RGD-PLUS. Cells can grow in 3D hydrogel at 1:1 and 1:3 dilutions of both hydrogels. Cells incorporate into the hydrogel in VitroGel 3D-RGD (A-D), but are not able to form network connections (E). However, in the new VitroGel RGD-PLUS, cells incorporate after 2 days (F-G), but they are able to form intercellular actin connections by day 7 (H-J).
Unlike the spheroid structure in VitroGel 3D-RGD, U-87 MG cells create better cell networking structures in VitroGel RGD-PLUS, indicating better cell-cell and cell-matrix interaction. These interactions are a crucial part of properly recapitulating in vivo tissue organization and regeneration and cancer in vitro. Although this is acknowledged, researchers have found it difficult to address, since there isn’t merely one solution to this problem. For example, in a standard in vivo cell niche, there are various types of cell interactions, shapes, junctions, etc., . Studies on currently existing 3D scaffolds have shown that the cohesive forces between cells, which drive the surface tension of the tissue and thus cell fate, can vary greatly between different parts of a tissue [56,57]. This can become problematic considering that some 3D matrices are more suitable to certain cell types and not others, introducing a certain level of variability into studies trying to use 3D methods of cell culture. This is evident even with our own studies. We see that different concentrations of the same matrix can lead to different cell behavior, even if cells are capable of growing in all concentrations.
The ability generates proper extracellular matrix interactions will also be a crucial part to properly understand the tumor microenvironment. A great deal of cancer progression is due to the ability of tumor cells to interact with their surroundings. For example, during tumor growth, the metastatic process begins with oxygen and nutrient deprivation, which triggers the release of angiogenic growth factors and cytokines that will cause blood vessel generation [58,59]. This nutrient and oxygen deprivation only happens after a certain threshold of growth that cannot be achieved in 2D culture, and which will require appropriate cell-cell and cell-matrix interaction in order to achieve in vivo modeling .
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Citation:Huang J (2019) 3D Cell Culture on VitroGel System. J Cytol Tissue Biol S1: 001.
Copyright: © 2019 John Huang, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.