Cell Culture Microenvironment Research Articles

Construction of a three-dimensional inflammation model of human bronchial epithelial cells BEAS-2B by using the self-assembling D-form peptide Sciobio-Ⅲ

Human bronchial epithelial cells in the airway system, as the primary barrier between humans and the surrounding environment, assume a crucial function in orchestrating the processes of airway inflammation. Target to develop a new three-dimensional (3D) inflammatory model to airway system, and here we report a strategy by using self-assembling D-form peptide to cover the process. By testing physicochemical properties and biocompatibility of Sciobio-Ⅲ, we confirmed that it can rapidly self-assembles under the trigger of ions to form a 3D nanonetwork-like scaffold, which supports 3D cell culture including the cell strains like BEAS-2B cells. Subsequently, inflammation model was established by lipopolysaccharide (LPS), the expression of some markers of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-8 (IL-8), the levels of relevant inflammatory factors were measured by RT-qPCR and the secretion profile of inflammatory cytokines by ELISA, are obtained the quite difference effects in 2D and 3D microenvironment, which suggested Sciobio-Ⅲ hydrogel is an ideal scaffold that create the microenvironment for 3D cell culture. Here we are success to establish a 3D inflammation model for airway system. This innovative model allows for rapid and accurate evaluation of drug metabolism and toxicological side effects, hope to use in drug screening for airway inflammatory diseases and beyond.

Towards the Standardization of Mesenchymal Stem Cell Secretome-Derived Product Manufacturing for Tissue Regeneration.

Mesenchymal stem cell secretome or conditioned medium (MSC-CM) is a combination of biomolecules and growth factors in cell culture growth medium, secreted by mesenchymal stem cells (MSCs), and the starting point of several derived products. MSC-CM and its derivatives could be applied after injuries and could mediate most of the beneficial regenerative effects of MSCs without the possible side effects of using MSCs themselves. However, before the clinical application of these promising biopharmaceuticals, several issues such as manufacturing protocols and quality control must be addressed. This review aims to underline the influence of the procedure for conditioned medium production on the quality of the secretome and its derivatives and highlights the questions considering cell sources and donors, cell expansion, cell passage number and confluency, conditioning period, cell culture medium, microenvironment cues, and secretome-derived product purification. A high degree of variability in MSC secretomes is revealed based on these parameters, confirming the need to standardize and optimize protocols. Understanding how bioprocessing and manufacturing conditions interact to determine the quantity, quality, and profile of MSC-CM is essential to the development of good manufacturing practice (GMP)-compliant procedures suitable for replacing mesenchymal stem cells in regenerative medicine.

A microfluidics-assisted photopolymerization method for high-resolution multimaterial 3D printing

3D printing and bioprinting are recognized as key technologies for the construction of complex micro-devices, micro-environments, and culture models. Thanks to their potential to produce precisely heterogeneous and 3D architectures, multimaterial printing methods, which enable the production of functional 3D structures integrating multiple materials, have attracted specific attention. Nevertheless, combining multimaterial and high-resolution printing is still a major challenge, and the available technologies do not provide simultaneously the resolution and multiplexing capabilities required to create heterogeneous 3D environments. In this work, we introduce the 3D-FlowPrint concept, which involves an opto-microfluidic printhead immersed in a liquid and moved above a surface. This technology combines the convenience of microfluidics in terms of the handling and delivery of small volumes of materials with the resolution provided by laser lithography. Delivered materials are hydrodynamically confined under the printhead owing to controlled aspiration of the injected material, ensuring a continuous supply of material and avoiding cross-contamination issues. Combining microfluidics with photo-polymerization provides unique advantages as it separates the polymerization process from the material delivery, permitting high-resolution polymerization (down to 10 μm) and multimaterial handling (switching time below 60 s). We present a first proof-of-concept using poly(ethylene glycol) diacrylate (PEGDA)-based hydrogels as a photosensitive material model, along with a detailed investigation of the influence of exposure parameters, printhead velocity, and hydrodynamic parameters on the fabrication of 2D and 3D heterogeneous structures. 3D-FlowPrint allows the creation of sub-millimetric to millimetric scale objects with multimaterial designs. A first validation was performed to show the potential of the approach in biology for the creation of engineered microenvironments for cell culture.

Differentiation of human spermatogonial stem cells using a human decellularized testicular scaffold supplemented by platelet-rich plasma.

Effective culture systems for attachment, migration, proliferation, and differentiation of spermatogonial stem cells (SSCs) can be a promising therapeutic modality for preserving male fertility. Decellularized extracellular matrix (ECM) from native testis tissue creates a local microenvironment for testicular cell culture. Furthermore, platelet-rich plasma (PRP) contains various growth factors for the proliferation and differentiation of SSCs. In this study, human testicular cells were isolated and cultured for 4 weeks, and SSCs were characterized using immunocytochemistry (ICC) and flow cytometry. Human testicular tissue was decellularized (0.3% SDS, 1% Triton), and the efficiency of the decellularization process was confirmed by histological staining and DNA content analysis. SSCs were cultured on the human decellularized testicular matrix (DTM) for 4 weeks. The viability and the expression of differentiation genes were evaluated by MTT and real-time polymerase chain reaction (PCR), respectively. Histological evaluation and DNA content analysis showed that the components of ECM were preserved during decellularization. Our results showed that after 4 weeks of culture, the expression levels of BAX, BCL-2, PLZF, and SCP3 were unchanged, while the expression of PRM2 significantly increased in the cells cultured on DTM supplemented with PRP (ECM-PRP). In addition, the expression of GFRA1 was significantly decreased in the ECM group compared to the control and PRP groups. Furthermore, the MTT test indicated that viability was significantly enhanced in cells plated on DTM supplemented with PRP. Our study demonstrated that DTM supplemented with PRP can provide an effective culture system for the differentiation and viability of SSCs.

Engineered cell culture microenvironments for mechanobiology studies of brain neural cells.

The biomechanical properties of the brain microenvironment, which is composed of different neural cell types, the extracellular matrix, and blood vessels, are critical for normal brain development and neural functioning. Stiffness, viscoelasticity and spatial organization of brain tissue modulate proliferation, migration, differentiation, and cell function. However, the mechanical aspects of the neural microenvironment are largely ignored in current cell culture systems. Considering the high promises of human induced pluripotent stem cell- (iPSC-) based models for disease modelling and new treatment development, and in light of the physiological relevance of neuromechanobiological features, applications of in vitro engineered neuronal microenvironments should be explored thoroughly to develop more representative in vitro brain models. In this context, recently developed biomaterials in combination with micro- and nanofabrication techniques 1) allow investigating how mechanical properties affect neural cell development and functioning; 2) enable optimal cell microenvironment engineering strategies to advance neural cell models; and 3) provide a quantitative tool to assess changes in the neuromechanobiological properties of the brain microenvironment induced by pathology. In this review, we discuss the biological and engineering aspects involved in studying neuromechanobiology within scaffold-free and scaffold-based 2D and 3D iPSC-based brain models and approaches employing primary lineages (neural/glial), cell lines and other stem cells. Finally, we discuss future experimental directions of engineered microenvironments in neuroscience.

A Porous Gelatin Methacrylate-Based Material for 3D Cell-Laden Constructs.

3D constructs are fundamental in tissue engineering and cancer modeling, generating a demand for tailored materials creating a suitable cell culture microenvironment and amenable to be bioprinted. Gelatin methacrylate (GelMA) is a well-known functionalized natural polymer with good printability and binding motifs allowing cell adhesion; however, its tight micropores induce encapsulated cells to retain a non-physiological spherical shape. To overcome this problem, blended GelMa is here blended with Pluronic F-127 (PLU) to modify the hydrogel internal porosity by inducing the formation of larger mesoscale pores. The change in porosity also leads to increased swelling and a slight decrease in Young's modulus. All blends form stable hydrogels both when cast in annular molds and bioprinted in complex structures. Embedded cells maintain high viability, and while Neuroblastoma cancer cells typically aggregate inside the mesoscale pores, Mesenchymal Stem Cells stretch in all three dimensions, forming cell-cell and cell-ECM interactions. The results of this work prove that the combination of tailored porous materials with bioprinting techniques enables to control both the micro and macro architecture of cell-laden constructs, a fundamental aspect for the development of clinically relevant in vitro constructs.

(Digital Presentation) Design, Flow Simulation and Experimental Test of Micro/Milibioreactor with Pillar Structures for Bubble Control and Cell Growth

The demand for Lab on a Chip (LOC) devices, such as microbioreactors, has driven microfluidic research and technology. With the capability of controlling the environment and monitoring results in real-time, microfluidic devices are highly advantageous in the application of cell culture. On the other hand, micro/nano-scale bubbles have become a field of growing interest for addressing many challenges in LOC applications. In this work, we present a novel microfluidic bioreactor based on a micropillars design that reduces stresses caused by bubbles on the cell growth in the microchip. The combination of one chamber with 3-pillars and 10-pillars design with the channel heights of 270 µm and 2 mm was evaluated.To obtain the optimal architecture, computational fluid dynamics (CFD) simulations using SolidWorks Flow Simulation Software were created to characterize the designs. Simulations are essential to assure that the washing of cells and bubbles do not occur in the fluid and to weeding out the designs that are suitable for cell growth. Numerical results showed that the velocity is high at the inlet and outlet when compared to the interior portions of the chamber. A higher flow rate and velocity were observed during the filling time in the 10-pillars design (3.0 x10-4 m/sec) in comparison with the 3-pillars design (2.4 x 10-4 m/sec). Also, a more homogeneous filling pattern was observed in the 3-pillars designs. This behavior occurs because the increased number of pillars manifests with a greater number of nuclei of flow disturbance, and more centers of agitation, resulting in a more heterogeneous flow. Pressure is homogenous for both 3 and 10 Pillars designs with slight changes from the inlet, with higher pressure, to the outlet, with lower pressure.The microfluidic devices were manufactured using flexographic master mold (Fmold) technology and polydimethylsiloxane (PDMS 10:1) as substrate. The total liquid capacity of the microchips was calculated as 205.5 μL for 270-um-height and 1522 μL for 2-mm-height. An experimental flow test with methylene blue using the syringe pump, with an inlet flow rate at 2.5 μL/sec, and image analysis from video using ImageJ software was performed. The experimental results were found to be comparable to the simulations results. When the number of pillars and height of the chambers increased, a higher flow rate and velocity were observed during the filling time. In this context, the 3-pillar design with 2 mm height was considered the most suitable design for cell culture applications. In addition, the 2 mm larger height design was tested for bubble control, and the results showed some of the bubbles were easily removed by applying flow rate, and the ones that remained inside the chamber did not block the channels in the pillar’s designs without affecting the system.The present platform has a simple architecture, inexpensive manufacturing methods, ease of operation, and the ability to use different types of cells. The pillar milli-bioreactor system has the advantages of bubble control, a large volume of work, real-time monitoring, and a suitable microenvironment for cell culture with prospects for future health applications.

Nucleoside-Derived Low-Molecular-Weight Gelators as a Synthetic Microenvironment for 3D Cell Culture.

For the last few decades, many efforts have been made in developing cell culture methods in order to overcome the biological limitations of the conventional two-dimensional culture. This paradigm shift is driven by a large amount of new hydrogel-based systems for three-dimensional culture, among other systems, since they are known to mimic some living tissue properties. One class of hydrogel precursors has received interest in the field of biomaterials, low-molecular-weight gelators (LMWGs). In comparison to polymer gels, LMWG gels are formed by weak interactions upon an external trigger between the molecular subunits, giving them the ability to reverse the gelation, thus showing potential for many applications of practical interest. This study presents the use of the nucleoside derivative subclass of LMWGs, which are glyco-nucleo-bola-amphiphiles, as a proof of concept of a 3D cell culture scaffold. Physicochemical characterization was performed in order to reach the optimal features to fulfill the requirements of the cell culture microenvironment, in terms of the mechanical properties, architecture, molecular diffusion, porosity, and experimental practicality. The retained conditions were tested by culturing glioblastoma cells for over a month. The cell viability, proliferation, and spatial organization showed during the experiments demonstrate the proof of concept of nucleoside-derived LMWGs as a soft 3D cell culture scaffold. One of the hydrogels tested permits cell proliferation and spheroidal organization over the entire culture time. These systems offer many advantages as they consume very few matters within the optimal range of viscoelasticity for cell culture, and the thermoreversibility of these hydrogels permits their use with few instruments. The LMWG-based scaffold for the 3D cell culture presented in this study unlocked the ability to grow spheroids from patient cells to reach personalized therapies by dramatically reducing the variability of the lattice used.

Melanoma growth in non-chemically modified translucid bacterial nanocellulose hollow and compartimentalized spheres

BackgroundBacterial nanocellulose (BNC) has been used as cell support in numerous tissue engineering studies. Its use can be explained based on the fact its structure allows the creation of a required microenvironment for an ideal material, which supports 3D cell culture. Its structure and interconnected pores lead to animal cells adhesion and proliferation, also allowing oxygen and nutrients transportation. MethodsWe developed a new methodology to produce spherical platforms synthesized by Komagataebacter hansenii (ATCC 23769) under dynamic culture conditions in minimal medium. The chemical composition and physical properties of the platforms were evaluated. Then, human melanoma cells (SK-MEL-28) were encapsulated into the platforms and evaluated by metabolic activity, morphology and their ability on adhering to the Hollow Translucid BNC Spheres (BNC-TS-H) and Compartmentalized Translucid BNC Spheres (BNC-TS-C) up to 3 days. ResultsBNC-TS-H and BNC-TS-C platforms were produced as translucid spheroid platforms with distinct microenvironment under dynamic fermentation. The chemical and physical characterizations confirmed the platforms composition as BNC. The produced internal microenvironments in spherical platforms are relevant to determine tumor cell fate. In the first 12 h of culture, cells could adhere to nanocellulose microfibers assuming their typical tumorous phenotype in 72 h of culture. ConclusionThe dynamic fermentation in minimal medium produced distinct microstructured platforms of BNC-TS-H and BNC-TS-C. The platforms microstructure resulted in microenvironments that enabled distinct cell-cell and cell-matrix interactions. This behavior suggests several applications in tissue engineering. General significanceThe method produced translucid BNC sphere platforms with distinct microenvironments for 3D cell culture.

Fabrication and Characterization of Sericin-PVA Composite Films from Gonometa postica, Gonometa rufobrunnea, and Argema mimosae: Potentially Applicable in Biomaterials.

This study deals with the fabrication and characterization of sericin-poly(vinyl alcohol) (PVA) composite films from three southern African silkworm cocoons. The sericin-PVA films were achieved by chemically cross-linking poly(vinyl alcohol) (PVA) with pure silk sericin protein using glutaraldehyde (GA) as a cross-linking agent. Fourier transform infrared (FTIR) results confirmed the overall cross-linking of pure silk sericin into PVA-GA networks to form cross-linked sericin-PVA films. This incident was shown by the incorporation of distinct major amide I (ν = 1640–1650 cm–1), amide II (ν = 1538–1540 cm–1), and amide III (ν = 1238–1244 cm–1) peaks. X-ray diffraction (XRD) showed sericin-PVA films to have two features, one representing amorphous and crystalline regions of silk sericin and the other representing sharp high-intensity PVA peaks at around 2θ = 20.2°, demonstrating a high crystallinity in the films as a result of the hydroxyl groups in its side chain. The swelling capacity of the three sericin-PVA films was influenced by the glutaraldehyde content used during the cross-linking process and pH of the aqueous medium into which the films were immersed after a period of time. The water contact angles of the sericin-PVA films were low, at 56.6 ± 0.56 and 60.2 ± 0.86, indicating further their hydrophilic nature. The scanning electron microscopy (SEM) images of the sericin-PVA films showed a rough texture with a granular network pattern on their surface. From the preliminary results, it was observed that the cytotoxicity of three sericin strains (Gonometa rufobrunnea, Argema mimosae, and Gonometa postica) had a cell viability percentage of 103, 90, and 80% respectively, demonstrating their biocompatibility in providing a favorable natural microenvironment for cell culture. The characterization results of the three silk sericin-PVA films demonstrated their potential for application in biomedical and biomaterial fields.

Inkjet‐Patterned Microdroplets as Individual Microenvironments for Adherent Single Cell Culture (Small 19/2022)

Single Cell Culture In article number 2107992, Jin-Ming Lin and co-workers culture adherent single cells in their individual microenvironments patterned by inkjet printing. Adhesion strength is confirmed by the in-situ microfluidic probe assay. Altered morphology among single cells features ultra-low aspect ratio and shows a distinct behavior caused by “singleness.”

Inkjet-Patterned Microdroplets as Individual Microenvironments for Adherent Single Cell Culture.

Adhesion of single cells is the foundation of manifold cellular behaviors and life processes. However, investigating the function of a specific cell is still challenging due to deficiency of adhesion or interference from surrounding cells. Herein, an open microfluidic system is reported for culturing adherent single cells, implemented by a micrometer-scale droplet matrix on an inkjet-printed polylysine template. The target cells are isolated from any cell from other droplets, and their adhesion strength is determined to be comparable to conventional petri dishes via an in-situ investigation with a microfluidic extractor. On this proposed platform, isolated single cells are observed to display an entirely distinct spreading behavior featuring total absence of elongation, indicating drastic cell behavior change from their "singleness." This system has high versatility and compatibility for various assaying methods, assuring a promising potential in detailed single cell behavior and cell heterogeneity studies.

Bioactive Decellularized Extracellular Matrix Hydrogel Microspheres Fabricated Using a Temperature-Controlling Microfluidic System.

Hydrogel microspheres have drawn great attention as functional three-dimensional (3D) microcarriers for cell attachment and growth, which have shown great potential in cell-based therapies and biomedical research. Hydrogels derived from a decellularized extracellular matrix (dECM) retain the intrinsic physical and biological cues from the native tissues, which often exhibit high bioactivity and tissue-specificity in promoting tissue regeneration. Herein, a novel two-stage temperature-controlling microfluidic system was developed which enabled production of pristine dECM hydrogel microspheres in a high-throughput manner. Porcine decellularized peripheral nerve matrix (pDNM) was used as the model raw dECM material for continuous generation of pDNM microgels without additional supporting materials or chemical crosslinking. The sizes of the microspheres were well-controlled by tuning the feed ratios of water/oil phases into the microfluidic device. The resulting pDNM microspheres (pDNM-MSs) were relatively stable, which maintained a spherical shape and a nanofibrous ultrastructure for at least 14 days. Schwann cells and PC12 cells preseeded on the pDNM-MSs not only showed excellent viability and an adhesive property, but also promoted cell extension compared to the commercially available gelatin microspheres. Moreover, primary neural stem/progenitor cells attached well to the pDNM-MSs, which further facilitated their proliferation. The successfully fabricated dECM hydrogel microspheres provided a highly bioactive microenvironment for 3D cell culture and functionalization, which showed promising potential in versatile biomedical applications.

Development of chitosan/hyaluronic acid hydrogel scaffolds via enzymatic reaction for cartilage tissue engineering

Biomimetic hydrogels have been developed in the fabrication of multifunctional microenvironments for cell culture and tissue fabrication. In the current study, different compositions of hyaluronic acid (HA) and chitosan (CH) derivatives with opposite charges were fabricated. To increase the interaction between these components phenol moieties were substituted on the backbone of HA (HAPH) and CH (CHPH) via carbodiimide-mediated condensation reaction, and then subjected to enzymatic cross-linking in the presence of horseradish peroxidase to create a stable hybrid microenvironment for cell encapsulation and tissue engineering. The gelation time, enzymatic degradation, and water contact angle of hydrogels reduced with increasing HAPH content. The rheological and mechanical properties of the hydrogels showed that a moderate concentration of HAPH can have the best results in the hydrogel structure. Hydrogel morphology altered depending on the amount of incorporated HA in CH precursor hydrogel solution and their pore size distribution decreased with increasing HAPH. The cellular studies showed proper cell viability and proliferation on optimum blend hydrogel surface compared with the neat hydrogels. Furthermore, the hybrid hydrogels demonstrated good characteristics for the expression of cartilage tissue markers and a higher propensity of MSCs to differentiate into cartilage-like cells compared to the control samples. Overall, the results suggest the optimum hybrid hydrogel can provide a superior biological microenvironment for chondrocytes in three-dimensional cartilage tissue engineering.

Nucleoside Derived Low Molecular Weight Gelators as a Synthetic Micro-Environment for 3D Cell Culture

Nucleoside Derived Low Molecular Weight Gelators as a Synthetic Micro-Environment for 3D Cell Culture

Encapsulation of murine hematopoietic stem and progenitor cells in a thiol-crosslinked maleimide-functionalized gelatin hydrogel

Biomaterial platforms are an integral part of stem cell biomanufacturing protocols. The collective biophysical, biochemical, and cellular cues of the stem cell niche microenvironment play an important role in regulating stem cell fate decisions. Three-dimensional (3D) culture of stem cells within biomaterials provides a route to present biophysical and biochemical stimuli through cell-matrix interactions and cell-cell interactions via secreted biomolecules. Herein, we describe a maleimide-functionalized gelatin (GelMAL) hydrogel that can be crosslinked via thiol-Michael addition click reaction for the encapsulation of sensitive stem cell populations. The maleimide functional units along the gelatin backbone enables gelation via the addition of a dithiol crosslinker without requiring external stimuli (e.g., UV light, photoinitiator), thereby reducing reactive oxide species generation. Additionally, the versatility of crosslinker selection enables easy insertion of thiol-containing bioactive or bioinert motifs. Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) were encapsulated in GelMAL, with mechanical properties tuned to mimic the in vivo bone marrow niche. We report the insertion of a cleavable peptide crosslinker that can be degraded by the proteolytic action of Sortase A, a mammalian-inert enzyme. Notably, Sortase A exposure preserves stem cell surface markers, which are an essential metric of hematopoietic activity used in immunophenotyping. This novel GelMAL system enables a route to produce artificial stem cell niches with tunable biophysical properties, intrinsic cell-interaction motifs, and orthogonal addition of bioactive crosslinks. Statement of significanceWe describe a maleimide-functionalized gelatin hydrogel that can be crosslinked via a thiol-maleimide mediated click reaction to form a stable hydrogel without the production of reactive oxygen species typical in light-based crosslinking. The mechanical properties can be tuned to match the in vivo bone marrow microenvironment for hematopoietic stem cell culture. Additionally, we report inclusion of a peptide crosslinker that can be cleaved via the proteolytic action of Sortase A and show that Sortase A exposure does not degrade sensitive surface marker expression patterns. Together, this approach reduces stem cell exposure to reactive oxygen species during hydrogel gelation and enables post-culture quantitative assessment of stem cell phenotype.

Assessing Various Control Samples for Microarray Gene Expression Profiling of Laryngeal Squamous Cell Carcinoma.

Selection of optimal control samples is crucial in expression profiling tumor samples. To address this issue, we performed microarray expression profiling of control samples routinely used in head and neck squamous cell carcinoma studies: human bronchial and tracheal epithelial cells, squamous cells obtained by laser uvulopalatoplasty and tumor surgical margins. We compared the results using multidimensional scaling and hierarchical clustering versus tumor samples and laryngeal squamous cell carcinoma cell lines. A general observation from our study is that the analyzed cohorts separated according to two dominant factors: “malignancy”, which separated controls from malignant samples and “cell culture-microenvironment” which reflected the differences between cultured and non-cultured samples. In conclusion, we advocate the use of cultured epithelial cells as controls for gene expression profiling of cancer cell lines. In contrast, comparisons of gene expression profiles of cancer cell lines versus surgical margin controls should be treated with caution, whereas fresh frozen surgical margins seem to be appropriate for gene expression profiling of tumor samples.

A Hydrogel Platform that Incorporates Laminin Isoforms for Efficient Presentation of Growth Factors – Neural Growth and Osteogenesis

AbstractLaminins (LMs) are important structural proteins of the extracellular matrix (ECM). The abundance of every LM isoform is tissue‐dependent, suggesting that LM has tissue‐specific roles. LM binds growth factors (GFs), which are powerful cytokines widely used in tissue engineering due to their ability to control stem cell differentiation. Currently, the most commonly used ECM mimetic material in vitro is Matrigel, a matrix of undefined composition containing LM and various GFs, but subjected to batch variability and lacking control of physicochemical properties. Inspired by Matrigel, a new and completely defined hydrogel platform based on hybrid LM‐poly(ethylene glycol) (PEG) hydrogels with controllable stiffness (1–25 kPa) and degradability is proposed. Different LM isoforms are used to bind and efficiently display GFs (here, bone morphogenetic protein (BMP‐2) and beta‐nerve growth factor (β‐NGF)), enabling their solid‐phase presentation at ultralow doses to specifically target a range of tissues. The potential of this platform to trigger stem cell differentiation toward osteogenic lineages and stimulate neural cells growth in 3D, is demonstrated. These hydrogels enable 3D, synthetic, defined composition, and reproducible cell culture microenvironments reflecting the complexity of the native ECM, where GFs in combination with LM isoforms yield the full diversity of cellular processes.

Aqueous Suspensions of Cellulose Oligomer Nanoribbons for Growth and Natural Filtration-Based Separation of Cancer Spheroids.

In vitro growth of cancer spheroids (CSs) and the subsequent separation of CSs from a 2D or 3D cell culture system are important for fundamental cancer studies and cancer drug screening. Although biopolymer-based or synthetic hydrogels are suitable candidates to be used as 3D cell culture scaffolds, alternatives with better processing capabilities are still required to set up cell culture microenvironment. In this study, we show that aqueous suspensions of crystalline nanoribbons composed of cellulose oligomers have a potential for CS growth and separation. The nanoribbon suspensions in serum-containing cell culture media fixed single cancer cells and CSs with large sizes in a 3D space, leading to suspension cultures for CS growth corresponding to culture time. Well-grown CSs were easily separated from the suspensions by natural filtration using a mesh filter with a suitable pore size. Cell viability tests revealed negligible cytotoxicity of the nanoribbons. In addition, physical damages to CSs by the separation procedures were negligible. Stable suspensions of biocompatible nanomaterials will thus provide novel microenvironments for growth and separation of diverse cell aggregates.

3D-printed Bioreactors for In Vitro Modeling and Analysis.

In recent years, three-dimensional (3D) printing has markedly enhanced the functionality of bioreactors by offering the capability of manufacturing intricate architectures, which changes the way of conducting in vitro biomodeling and bioanalysis. As 3D-printing technologies become increasingly mature, the architecture of 3D-printed bioreactors can be tailored to specific applications using different printing approaches to create an optimal environment for bioreactions. Multiple functional components have been combined into a single bioreactor fabricated by 3D-printing, and this fully functional integrated bioreactor outperforms traditional methods. Notably, several 3D-printed bioreactors systems have demonstrated improved performance in tissue engineering and drug screening due to their 3D cell culture microenvironment with precise spatial control and biological compatibility. Moreover, many microbial bioreactors have also been proposed to address the problems concerning pathogen detection, biofouling, and diagnosis of infectious diseases. This review offers a reasonably comprehensive review of 3D-printed bioreactors for in vitro biological applications. We compare the functions of bioreactors fabricated by various 3D-printing modalities and highlight the benefit of 3D-printed bioreactors compared to traditional methods.