Conventional Cell Culture Techniques Research Articles

Edible mycelium as proliferation and differentiation support for anchorage-dependent animal cells in cultivated meat production

Cultivated meat production requires bioprocess optimization to achieve cell densities that are multiple orders of magnitude higher compared to conventional cell culture techniques. These processes must maximize resource efficiency and cost-effectiveness by attaining high cell growth productivity per unit of medium. Microcarriers, or carriers, are compatible with large-scale bioreactor use, and offer a large surface-area-to-volume ratio for the adhesion and proliferation of anchorage-dependent animal cells. An ongoing challenge persists in the efficient retrieval of cells from the carriers, with conflicting reports on the effectiveness of trypsinization and the need for additional optimization measures such as carrier sieving. To surmount this issue, edible carriers have been proposed, offering the advantage of integration into the final food product while providing opportunities for texture, flavor, and nutritional incorporation. Recently, a proof of concept (POC) utilizing inactivated mycelium biomass derived from edible filamentous fungus demonstrated its potential as a support structure for myoblasts. However, this POC relied on a model mammalian cell line combination with a single mycelium species, limiting realistic applicability to cultivated meat production. This study aims to advance the POC. We found that the species of fungi composing the carriers impacts C2C12 myoblast cell attachment—with carriers derived from Aspergillus oryzae promoting the best proliferation. C2C12 myoblasts effectively differentiated on mycelium carriers when induced in myogenic differentiation media. Mycelium carriers also supported proliferation and differentiation of bovine satellite cells. These findings demonstrate the potential of edible mycelium carrier technology to be readily adapted in product development within the cultivated meat industry.

Evaluation of cigarette smoke-induced oxidative stress and inflammation in BEAS-2B cells based on a lung microfluidic chip.

Oxidative stress and inflammation induced by cigarette smoking are associated with the pathology process of various chronic respiratory diseases, including asthma, emphysema, chronic obstructive pulmonary disease and cancer. Compared with conventional cell culture techniques, microfluidic chips can provide a continuous nutrient supply, mimic the in vivo physiological microenvironment of the cells, and conduct an integrated and flexible analysis of cell status and functions. Here, we designed and fabricated a bionic-lung chip, which was applied to perform cigarette smoke exposure of BEAS-2B cells cultured at the gas-liquid interface. The oxidative stress and inflammation in the cells exposed to cigarette smoke were investigated on chip. The results showed that cellular damage, oxidative stress and inflammatory response induced by cigarette smoke in the chip were dependent on smoke concentration and time after smoke exposure. N-Acetylcysteine (NAC) significantly inhibited these effects of cigarette smoke exposure on the cells at the gas-liquid interface within the chip.

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall.

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Modeling-based design specifications for microfluidic gradients generators for biomedical applications

This work proposes a comprehensive approach to optimize the design of microfluidic concentration gradient generators (MGGs) for biomedical applications. Exposing biological systems to controlled gradients enables fast screenings of induced concentration-dependent signals and surpasses several limitations of conventional cell culture techniques. The MGG working principle is the formation of diffusion-driven concentration gradients between a source and a sink, both connected to a cell culture chamber through an array of microchannels. The devices were modeled with Comsol Multiphysics®, in a combined fluid-dynamic and mass-transport study allowing prediction of the internal fields and guiding design optimization. Ideal MGGs must ensure fast transients (< 1 h) to reach a steady gradient. To identify the key features determining the device performance, we analyzed design specifications and operating parameters including: the shape of the source and sink (width 1–2 mm), the number and length of the microchannels (17–34, and 2.5–5 mm), the flow rate (5–10 μl/min). Our results prove that the formation and shape of the gradient are strongly affected by the device geometry, and mostly by the microchannels length. In addition, higher flow rates lead to the generation of stagnation zones and increase the gradient steepness. The model optimized MGG was fabricated and proved successful in the generation of stable concentration gradients over cell monolayers inside the culture chamber.

Skin-on-a-Chip Technology: Microengineering Physiologically Relevant In Vitro Skin Models.

The increased demand for physiologically relevant in vitro human skin models for testing pharmaceutical drugs has led to significant advancements in skin engineering. One of the most promising approaches is the use of in vitro microfluidic systems to generate advanced skin models, commonly known as skin-on-a-chip (SoC) devices. These devices allow the simulation of key mechanical, functional and structural features of the human skin, better mimicking the native microenvironment. Importantly, contrary to conventional cell culture techniques, SoC devices can perfuse the skin tissue, either by the inclusion of perfusable lumens or by the use of microfluidic channels acting as engineered vasculature. Moreover, integrating sensors on the SoC device allows real-time, non-destructive monitoring of skin function and the effect of topically and systemically applied drugs. In this Review, the major challenges and key prerequisites for the creation of physiologically relevant SoC devices for drug testing are considered. Technical (e.g., SoC fabrication and sensor integration) and biological (e.g., cell sourcing and scaffold materials) aspects are discussed. Recent advancements in SoC devices are here presented, and their main achievements and drawbacks are compared and discussed. Finally, this review highlights the current challenges that need to be overcome for the clinical translation of SoC devices.

Preparation and Cultivation of Colonic and Small Intestinal Murine Organoids Including Analysis of Gene Expression and Organoid Viability.

Organoids are complex three-dimensional structures, which contain different cell types and help to overcome many limitations of conventional 2D cell culture techniques. Here, we present a protocol for the cultivation of murine matched-pairs of small intestinal and colonic epithelial organoids, and colonic tumor organoids derived from the chemical colorectal cancer (CRC) AOM/DSS mouse model. Therefore, intestinal crypts or tumor tissue containing stem cells are isolated from the same donor mouse and cultivated in Matrigel®. The culture medium is supplemented with different growth factors to model the intestinal stem cell niche, allowing their self-renewal and differentiation. Matched-pair organoids enable the analysis of pharmacological effects and the tumor selectivity of drugs. Graphic abstract: Schematic overview of colonic matched pair organoid preparation, generated from the chemical AOM/DSS colorectal cancer mouse model. Please note that normal colon-derived organoids (green) differ in their morphology from tumor-derived organoids (red). Normal colonic-derived organoids display a thicker and crypt-like epithelial layer, whereas tumor-derived organoids are round with a thin epithelial layer.

Analysis of the influence of inner morphology on blood flow in 3D-printed bone scaffolds

In recent years, 3D printed scaffolds have been proposed as promising alternative to the conventional cell culture techniques. Scaffolds, indeed, allow the development of a higher number of cellular connections along the three dimensions favoring the cell regeneration, which make them particularly suitable in case of implants for deteriorate bones in old age patients. Besides the characteristics of biocompatibility and biodegradability fundamental for the integration of the scaffolds with the human body, the inner morphology, the permeability as well as the porosity are parameters of paramount relevance in the design of 3D-printed scaffolds influencing the flow of the blood through the cells and, thus, their metabolic functions. In the present work the influence of the internal geometry of 3D-printed scaffolds on the blood flow was investigated. Five cylindrical scaffolds having different internal geometry and different porosity were fabricated using parametric design technique. Numerical analysis of the blood flow within the designed structures was conducted by using CFD tool.

Hepatitis A related to foods

Hepatitis A is caused by hepatitis A virus (HAV). Hepatitis A virus (HAV) is classified as a picornavirus. Hepatitis A virus infection occurs globally and is causing a public health concern, primarily in developing countries due to its persistent circulation in the environment. The improved sanitary condition and increase in awareness of personal hygiene have led to the marked reduction of HAV prevalence in industrialized countries during childhood and to a shift of the infection towards adulthood. HAV is an environmentally stable, positive single stranded RNA virus that is primarily transmitted by the fecal-oral route, person to person contact or ingestion of contaminated food and drink. One of the main causes leading to HAV infection is epidemiologically linked to the consumption of raw or undercooked shellfish particularly oysters and clams. Due to their filter-feeding style, these shellfishes readily concentrate viruses from the surrounding water containing municipal sewage, and as a consequence pose a health threat to consumers. Therefore, development of detection techniques possessing the requisite sensitivity and specificity for the practical routine monitoring purposes is of great importance necessary for the protection of shellfish-consuming public. Nucleic acid based method such as reverse transcription PCR has emerged as the popular method of choice in view of rapidity, accuracy and sensitivity in contrary of the time-consuming conventional cell culture and hybridization techniques. The low concentration of viral genome present in the environmental sample which requires effective isolation and concentration of virions and the labor-extensive purification, removal of PCR inhibitors will be unfavorable

Electrophysiology Read-Out Tools for Brain-on-Chip Biotechnology.

Brain-on-Chip (BoC) biotechnology is emerging as a promising tool for biomedical and pharmaceutical research applied to the neurosciences. At the convergence between lab-on-chip and cell biology, BoC couples in vitro three-dimensional brain-like systems to an engineered microfluidics platform designed to provide an in vivo-like extrinsic microenvironment with the aim of replicating tissue- or organ-level physiological functions. BoC therefore offers the advantage of an in vitro reproduction of brain structures that is more faithful to the native correlate than what is obtained with conventional cell culture techniques. As brain function ultimately results in the generation of electrical signals, electrophysiology techniques are paramount for studying brain activity in health and disease. However, as BoC is still in its infancy, the availability of combined BoC–electrophysiology platforms is still limited. Here, we summarize the available biological substrates for BoC, starting with a historical perspective. We then describe the available tools enabling BoC electrophysiology studies, detailing their fabrication process and technical features, along with their advantages and limitations. We discuss the current and future applications of BoC electrophysiology, also expanding to complementary approaches. We conclude with an evaluation of the potential translational applications and prospective technology developments.

Microfluidic perfusion modulates growth and motor neuron differentiation of stem cell aggregates.

Microfluidic technologies provide many advantages for studying differentiation of three-dimensional (3D) stem cell aggregates, including the ability to control the culture microenvironment, isolate individual aggregates for longitudinal tracking, and perform imaging-based assays. However, applying microfluidics to studying mechanisms of stem cell differentiation requires an understanding of how microfluidic culture conditions impact cell phenotypes. Conventional cell culture techniques cannot directly be applied to the microscale, as microscale culture varies from macroscale culture in multiple aspects. Therefore, the objective of this work was to explore key parameters in microfluidic culture of 3D stem cell aggregates and to understand how these parameters influence stem cell behavior and differentiation. These studies were done in the context of differentiation of embryonic stem cells (ESCs) to motor neurons (MNs). We assessed how media exchange frequency modulates the biochemical microenvironment, including availability of exogenous factors (e.g. nutrients, small molecule additives) and cell-secreted molecules, and thereby impacts differentiation. The results of these studies provide guidance on how key characteristics of 3D cell cultures can be considered when designing microfluidic culture parameters. We demonstrate that discontinuous perfusion is effective at supporting stem cell aggregate growth. We find that there is a balance between the frequency of media exchange, which is needed to ensure that cells are not nutrient-limited, and the need to allow accumulation of cell-secreted factors to promote differentiation. Finally, we show how microfluidic device geometries can influence transport of biomolecules and potentially promote asymmetric spatial differentiation. These findings are instructive for future work in designing devices and experiments for culture of cell aggregates.

Recent Advances in Engineering the Stem Cell Microniche in 3D.

Conventional 2D cell culture techniques have provided fundamental insights into key biochemical and biophysical mechanisms responsible for various cellular behaviors, such as cell adhesion, spreading, division, proliferation, and differentiation. However, 2D culture in vitro does not fully capture the physical and chemical properties of the native microenvironment. There is a growing body of research that suggests that cells cultured on 2D substrates differ greatly from those grown in vivo. This article focuses on recent progress in using bioinspired 3D matrices that recapitulate as many aspects of the natural extracellular matrix as possible. A range of techniques for the engineering of 3D microenvironment with precisely controlled biophysical and chemical properties, and the impact of these environments on cellular behavior, is reviewed. Finally, an outlook on future challenges for engineering the 3D microenvironment and how such approaches would further our understanding of the influence of the microenvironment on cell function is provided.

Bacterial Cellulose Shifts Transcriptome and Proteome of Cultured Endothelial Cells Towards Native Differentiation

Preserving the native phenotype of primary cells in vitro is a complex challenge. Recently, hydrogel-based cellular matrices have evolved as alternatives to conventional cell culture techniques. We developed a bacterial cellulose-based aqueous gel-like biomaterial, dubbed Xellulin, which mimics a cellular microenvironment and seems to maintain the native phenotype of cultured and primary cells. When applied to human umbilical vein endothelial cells (HUVEC), it allowed the continuous cultivation of cell monolayers for more than one year without degradation or dedifferentiation. To investigate the impact of Xellulin on the endothelial cell phenotype in detail, we applied quantitative transcriptomics and proteomics and compared the molecular makeup of native HUVEC, HUVEC on collagen-coated Xellulin and collagen-coated cell culture plastic (polystyrene).Statistical analysis of 12,475 transcripts and 7831 proteins unveiled massive quantitative differences of the compared transcriptomes and proteomes. K-means clustering followed by network analysis showed that HUVEC on plastic upregulate transcripts and proteins controlling proliferation, cell cycle and protein biosynthesis. In contrast, HUVEC on Xellulin maintained, by and large, the expression levels of genes supporting their native biological functions and signaling networks such as integrin, receptor tyrosine kinase MAP/ERK and PI3K signaling pathways, while decreasing the expression of proliferation associated proteins. Moreover, CD34-an endothelial cell differentiation marker usually lost early during cell culture - was re-expressed within 2 weeks on Xellulin but not on plastic. And HUVEC on Xellulin showed a significantly stronger functional responsiveness to a prototypic pro-inflammatory stimulus than HUVEC on plastic.Taken together, this is one of the most comprehensive transcriptomic and proteomic studies of native and propagated HUVEC, which underscores the importance of the morphology of the cellular microenvironment to regulate cellular differentiation, and demonstrates, for the first time, the potential of Xellulin as versatile tool promoting an in vivo-like phenotype in primary and propagated cell culture.

Engineering Cellular Microenvironments with Photo- and Enzymatically Responsive Hydrogels: Toward Biomimetic 3D Cell Culture Models.

Conventional cell culture techniques using 2D polystyrene or glass have provided great insight into key biochemical mechanisms responsible for cellular events such as cell proliferation, differentiation, and cell-cell interactions. However, the physical and chemical properties of 2D culture in vitro are dramatically different than those found in the native cellular microenvironment in vivo. Cells grown on 2D substrates differ significantly from those grown in vivo, and this explains, in part, why many promising drug candidates discovered through in vitro drug screening assays fail when they are translated to in vivo animal or human models. To overcome this obstacle, 3D cell culture using biomimetic hydrogels has emerged as an alternative strategy to recapitulate native cell growth in vitro. Hydrogels, which are water-swollen polymers, can be synthetic or naturally derived. Many methods have been developed to control the physical and chemical properties of the hydrogels to match those found in specific tissues. Compared to 2D culture, cells cultured in 3D gels with the appropriate physicochemical cues can behave more like they naturally do in vivo. While conventional hydrogels involve modifications to the bulk material to mimic the static aspects of the cellular microenvironment, recent progress has focused on using more dynamic hydrogels, the chemical and physical properties of which can be altered with external stimuli to better mimic the dynamics of the native cellular microenvironment found in vivo. In this Account, we describe our progress in designing stimuli-responsive, optically transparent hydrogels that can be used as biomimetic extracellular matrices (ECMs) to study cell differentiation and migration in the context of modeling the nervous system and cancer. Specifically, we developed photosensitive agarose and hyaluronic acid hydrogels that are activated by single or two-photon irradiation for biomolecule immobilization at specific volumes within the 3D hydrogel. By controlling the spatial location of protein immobilization, we created 3D patterns and protein concentration gradients within these gels. We used the latter to study the effect of VEGF-165 concentration gradients on the interactions between endothelial cells and retinal stem cells. Hyaluronic acid (HA) is particularly compelling as it is naturally found in the ECM of many tissues and the tumor microenvironment. We used Diels-Alder click chemistry and cryogelation to alter the chemical and physical properties of these hydrogels. We also designed HA hydrogels to study the invasion of breast cancer cells. HA gels were chemically cross-linked with matrix metalloproteinase (MMP)-degradable peptides that degrade in the presence of cancer cell-secreted MMPs, thus allowing cells to remodel their local microenvironment and invade into HA/MMP-degradable gels.

3D Hanging Drop Culture to Establish Prostate Cancer Organoids.

Three-dimensional (3D) cell culture enables the growth of cells in a multidimensional and multicellular manner compared to conventional cell culture techniques. Especially in prostate cancer research there is a big need for more tissue-recapitulating models to get a better understanding of the mechanisms driving prostate cancer as well as to screen for more efficient drugs that can be used for treatment. In this chapter we describe a 3D hanging drop system that can be used to culture prostate cancer organoids as tumor epithelial monocultures and as epithelial-stromal cocultures.

Isolation of Zika Virus Imported from Tonga into Australia.

Introduction: The globally emergent Zika virus (ZIKV) is a threat to Australia, given the number of imported cases from epidemic regions and the presence of competent mosquito vectors. We report the isolation of ZIKV from a female traveler who recently returned from Tonga to Brisbane, Queensland, Australia in 2016.Methods: A specific TaqMan real-time reverse transcriptase polymerase chain reaction assay (RT-PCR) assay was used to detect ZIKV in serum and urine samples. Conventional cell culture techniques and suckling mice were employed in an attempt to isolate ZIKV from serum and urine.Results: A ZIKV isolate (TS17-2016) was recovered from the serum sample after one passage in suckling mouse brains and harvested 11 days post inoculation. Phylogenetic analysis of complete envelope (E) gene sequences demonstrated TS17-2016 shared 99.9% nucleotide identity with other contemporary sequences from Tonga 2016, Brazil 2015 and French Polynesia 2013 within the Asian lineage.Discussion: This is the first known report of successful isolation of ZIKV from a human clinical sample in Australia and the first from a traveler from Tonga. This study highlights the potential difficulties in isolating ZIKV from acute clinical samples using conventional cell culture techniques, particularly in non-endemic countries like Australia where access to samples of sufficient viral load is limited. The successful isolation of TS17-2016 will be essential for continued investigations of ZIKV transmission and pathogenicity and will enable the advancement of new preventative control measures extremely relevant to the Australian and Pacific region.

An open-chamber flow-focusing device for focal stimulation of micropatterned cells

Microfluidic devices can deliver soluble factors to cell and tissue culture microenvironments with precise spatiotemporal control. However, enclosed microfluidic environments often have drawbacks such as the need for continuous culture medium perfusion which limits the duration of experiments, incongruity between microculture and macroculture, difficulty in introducing cells and tissues, and high shear stress on cells. Here, we present an open-chamber microfluidic device that delivers hydrodynamically focused streams of soluble reagents to cells over long time periods (i.e., several hours). We demonstrate the advantage of the open chamber by using conventional cell culture techniques to induce the differentiation of myoblasts into myotubes, a process that occurs in 7-10 days and is difficult to achieve in closed chamber microfluidic devices. By controlling the flow rates and altering the device geometry, we produced sharp focal streams with widths ranging from 36 μm to 187 μm. The focal streams were reproducible (∼12% variation between units) and stable (∼20% increase in stream width over 10 h of operation). Furthermore, we integrated trenches for micropatterning myoblasts and microtraps for confining single primary myofibers into the device. We demonstrate with finite element method (FEM) simulations that shear stresses within the cell trench are well below values known to be deleterious to cells, while local concentrations are maintained at ∼22% of the input concentration. Finally, we demonstrated focused delivery of cytoplasmic and nuclear dyes to micropatterned myoblasts and myofibers. The open-chamber microfluidic flow-focusing concept combined with micropatterning may be generalized to other microfluidic applications that require stringent long-term cell culture conditions.

Placenta-on-a-chip: a novel platform to study the biology of the human placenta

Objective: Studying the biology of the human placenta represents a major experimental challenge. Although conventional cell culture techniques have been used to study different types of placenta-derived cells, current in vitro models have limitations in recapitulating organ-specific structure and key physiological functions of the placenta. Here we demonstrate that it is possible to leverage microfluidic and microfabrication technologies to develop a microengineered biomimetic model that replicates the architecture and function of the placenta.Materials and methods: A “Placenta-on-a-Chip” microdevice was created by using a set of soft elastomer-based microfabrication techniques known as soft lithography. This microsystem consisted of two polydimethylsiloxane (PDMS) microfluidic channels separated by a thin extracellular matrix (ECM) membrane. To reproduce the placental barrier in this model, human trophoblasts (JEG-3) and human umbilical vein endothelial cells (HUVECs) were seeded onto the opposite sides of the ECM membrane and cultured under dynamic flow conditions to form confluent epithelial and endothelial layers in close apposition. We tested the physiological function of the microengineered placental barrier by measuring glucose transport across the trophoblast-endothelial interface over time. The permeability of the barrier study was analyzed and compared to that obtained from acellular devices and additional control groups that contained epithelial or endothelial layers alone.Results: Our microfluidic cell culture system provided a tightly controlled fluidic environment conducive to the proliferation and maintenance of JEG-3 trophoblasts and HUVECs on the ECM scaffold. Prolonged culture in this model produced confluent cellular monolayers on the intervening membrane that together formed the placental barrier. This in vivo-like microarchitecture was also critical for creating a physiologically relevant effective barrier to glucose transport. Quantitative investigation of barrier function was conducted by calculating permeability coefficients and metabolic rates in varying conditions of barrier structure. The rates of glucose transport and metabolism were consistent with previously reported in vivo observations.Conclusion: The “Placenta-on-a-Chip” microdevice described herein provides new opportunities to simulate and analyze critical physiological responses of the placental barrier. This system may be used to address the major limitations of existing placenta model systems and serve to enable research platforms for reproductive biology and medicine.

Biofabrication of tissue constructs by 3D bioprinting of cell-laden microcarriers

Bioprinting allows the fabrication of living constructs with custom-made architectures by spatially controlled deposition of multiple bioinks. This is important for the generation of tissue, such as osteochondral tissue, which displays a zonal composition in the cartilage domain supported by the underlying subchondral bone. Challenges in fabricating functional grafts of clinically relevant size include the incorporation of cues to guide specific cell differentiation and the generation of sufficient cells, which is hard to obtain with conventional cell culture techniques. A novel strategy to address these demands is to combine bioprinting with microcarrier technology. This technology allows for the extensive expansion of cells, while they form multi-cellular aggregates, and their phenotype can be controlled. In this work, living constructs were fabricated via bioprinting of cell-laden microcarriers. Mesenchymal stromal cell (MSC)-laden polylactic acid microcarriers, obtained via static culture or spinner flask expansion, were encapsulated in gelatin methacrylamide-gellan gum bioinks, and the printability of the composite material was studied. This bioprinting approach allowed for the fabrication of constructs with high cell concentration and viability. Microcarrier encapsulation improved the compressive modulus of the hydrogel constructs, facilitated cell adhesion, and supported osteogenic differentiation and bone matrix deposition by MSCs. Bilayered osteochondral models were fabricated using microcarrier-laden bioink for the bone compartment. These findings underscore the potential of this new microcarrier-based biofabrication approach for bone and osteochondral constructs.

Effect of 3D Matrix Compositions on the Efficacy of EGFR Inhibition in Pancreatic Ductal Adenocarcinoma Cells

Therapeutics to inhibit signaling of epidermal growth factor receptor (EGFR) has been suggested as a potential treatment for pancreatic cancers, and two-dimensional (2D) cell culture techniques are commonly used to identify and/or verify the therapeutic efficacy of EGFR inhibitors. However, drug targets identified from conventional cell culture techniques may not exhibit desired functions when these drugs are tested in animal studies, in large part due to the complicated tumor microenvironments. Hence, it is crucial to develop a biomimetic cell culture system capable of recapitulating aspects of tumor niches for studying cancer cell fate processes under the influence of various environmental stimuli. In this study, we utilized a versatile PEG-peptide hydrogel system to demonstrate the influence of matrix properties and EGFR inhibition on the growth of a pancreatic ductal adenocarcinoma cell line (PANC-1). PANC-1 cells were encapsulated in 8-arm PEG-norbornene (PEG8NB) hydrogels cross-linked by matrix metalloproteinase (MMP) sensitive peptide (MMP(Linker)) using thiol-ene photoclick chemistry. In soft hydrogels (G' ~ 2 kPa), cells retained high initial viability and formed clusters after prolonged culture, whereas cells encapsulated in stiff hydrogels (G' ~ 12 kPa) exhibited lower initial viability and reduced proliferation. While the immobilization of an EGFR peptide inhibitor, Asn-Tyr-Gln-Gln-Asn or NYQQN, in soft hydrogels did not cause cell death, this peptide induced significant cell apoptosis when immobilized in stiff hydrogels. Western blotting results showed that cell death was due to reduced expression of EGFR and Akt in stiff hydrogels under the influence of immobilized NYQQN peptide. These results shed light on the importance and non-negligible role of matrix properties on the efficacy of antitumor drugs.

Multicellular Tumor Spheroids as an in Vivo–Like Tumor Model for Three-Dimensional Imaging of Chemotherapeutic and Nano Material Cellular Penetration

We present a flexible and highly reproducible method using three-dimensional (3D) multicellular tumor spheroids to quantify chemotherapeutic and nanoparticle penetration properties in vitro. We generated HeLa cell-derived spheroids using the liquid overlay method. To properly characterize HeLa spheroids, scanning electron microscopy, transmission electron microscopy, and multiphoton microscopy were used to obtain high-resolution 3D images of HeLa spheroids. Next, pairing high-resolution optical characterization techniques with flow cytometry, we quantitatively compared the penetration of doxorubicin, quantum dots, and synthetic micelles into 3D HeLa spheroid versus HeLa cells grown in a traditional two-dimensional culturing system. Our data revealed that 3D cultured HeLa cells acquired several clinically relevant morphologic and cellular characteristics (such as resistance to chemotherapeutics) often found in human solid tumors. These characteristic, however, could not be captured using conventional two-dimensional cell culture techniques. This study demonstrated the remarkable versatility of HeLa spheroid 3D imaging. In addition, our results revealed the capability of HeLa spheroids to function as a screening tool for nanoparticles or synthetic micelles that, due to their inherent size, charge, and hydrophobicity, can penetrate into solid tumors and act as delivery vehicles for chemotherapeutics. The development of this image-based, reproducible, and quantifiable in vitro HeLa spheroid screening tool will greatly aid future exploration of chemotherapeutics and nanoparticle delivery into solid tumors.