3D cell culture

Background: Topical drug administration offers an attractive route with minimal invasiveness. It also avoids limitations of intravenous administration such as the first pass metabolism and presystemic elimination within the gastrointestinal tract. Furthermore, topical drug administration is safe, have few side effects, is easy to apply, and offers a fast onset of action. However, the development of effective topical formulations still represents a challenge for the desired effect to be reached, locally or systemically. Solid lipid nanoparticles and nanos-tructured lipid carriers are particular candidates to overcome the problem of topical drug administration. The nanometric particle size of lipid nanoparticles favors the physical adhesion to the skin or mucosal, what can also be attained with the formation of hybrid (nanoparticles/polymer) systems. Methods: In this review, we discuss the major challenges for lipid nanoparticles formulations for topical application to oral mucosa, skin, and eye, highlighting the strategies to improve the performance of lipid nanoparticles for topical applications. Next, we critically analyzed the in vitro and in vivo approaches used to evaluate lipid nanoparticles performance and toxicity. Conclusion: We addressed some major drawbacks related to lipid nanoparticle topical formulations and concluded the key points that have to be overcome to help them to reach the market in topical formulations to oral mucosa, skin, and eye.

Hydrogels are prototypical matrices for 3D cell culture, of which alginate hydrogels are extensively used. However, ionic crosslinking agents, such as Ca 2+ , are required to form alginate hydrogels, but introduce Ca 2+-associated cytotoxicity and long-term stability issues. Collagen is a promising biomaterial for 3D cell culture scaffolds primarily due to its biocompatibility. In the present study, the authors designed and fabricated a calcium-free, physically crosslinked, efficient, and bioactive hydrogel composed of alginate, marine collagen, and agarose (AmCA) for use in 3D cell cultures. This AmCA hydrogel was assessed by FTIR, swelling property, scanning electron microscopy, phase contrast microscopy, cell proliferation, cell viability, confocal microscopy, transparency and RT-PCR analyses. The gel was found to exhibit excellent cytocompatibility with various tumor and non-tumor cells, to generate high yields of multicellular spheroids, and to promote cellular activity. Furthermore, the transparency of the AmCA hydrogel suggests it can be used without cell-tracking chemicals in morphological studies of cell cultures. Taken together, it would appear that the described physically crosslinked AmCA hydrogel could provide a novel platform for the development of customizable, transparent, biocompatible, functional, easy-to-produce, and cost-effective scaffolds for use in 3D cultures of various cell types.

Bone tissue engineering, using a porous scaffold material to induce the formation of bone from the surrounding
tissues has some distinct advantages over autografting and allografting, and it is a rapidly growing alternative
approach to heal damaged bone tissue. The current study focuses on fabrication and characterization of
hydroxyapatite and natural biopolymer composite for improved bone repair and regeneration. The porous composite
scaffold containing hydroxyapatite (HAp), chitosan and gelatin having 3D network of interconnected pores with an
average pore size of 150 m was fabricated using freeze drying method. Glutaraldehyde was used as a cross linker
between chitosan and gelatin. Characteristic IR band for (–C=N–) group observed in IR study of the prepared
scaffold indicated the crosslinking ability of glutaraldehyde with chitosan and gelatin. Total porosity in the scaffold
varied between 60 and 70% as determined by Archimedes principle and depended on the solids loading of the
scaffold. Microstructural analysis using field emission scanning electron microscopy (FESEM) showed an
interconnected porous network in the scaffold where HAp nanoparticles were found to be dispersed in the biopolymer
matrix. Thick bone like apatite layer deposition was observed on the surface of the scaffold on immersing it into
simulated body fluid (SBF) at 37oC for 7 days. The developed scaffold exhibited a compressive strength of 1.75
MPa which is close to the lower limit of compressive strength of spongy bone. These types of composite scaffolds
find application as efficient biomaterial for natural bone tissue regeneration and bone tissue engineering.

Alongside cancer cells, tumours exhibit a complex stroma containing a repertoire of cells, matrix molecules and soluble factors that actively crosstalk between each other. Recognition of this multifaceted concept of the tumour microenvironment (TME) calls for authentic TME mimetics to study cancer in vitro. Traditionally, tumourigenesis has been investigated in non-human, three-dimensional rat type I collagen containing organotypic discs or by means of mouse sarcoma-derived gel, such as Matrigel w. However, the molecular compositions of these simplified assays do not properly simulate human TME. Here, we review the main properties and benefits of using human leiomyoma discs and their matrix Myogel for in vitro assays. Myoma discs are practical for investigating the invasion of cancer cells, as are cocultures of cancer and stromal cells in a stiff, hypoxic TME mimetic. Myoma discs contain soluble factors and matrix molecules commonly present in neoplastic stroma. In Transwell, IncuCyte, spheroid and sandwich assays, cancer cells move faster and form larger colonies in Myogel than in Matrigel w. Additionally, Myogel can replace Matrigel w in hanging-drop and tube-formation assays. Myogel also suits three-dimensional drug testing and extracellular vesicle interactions. To conclude, we describe the application of our myoma-derived matrices in 3D in vitro cancer assays.

Inflammatory episodes observed in multiple sclerosis are characterized by perivascular infiltration of central nervous system (CNS) tissue by circulating leucocytes directed by chemokines and activation of both microglia and astrocytes. An in vitro approach, using a three-dimensional aggregate culture system derived from foetal rat brain tissue, is being taken to study the effect of inflammatory mediators on CNS cells. These aggregates model the brain in vivo in terms of glial and neuronal development and cell-cell interactions. Aggregates cultures were treated with lipopolysaccharide (LPS) and the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFNγ). The effect of these treatments on the expression of a range of chemokine receptors (CCR1, CCR2, CCR5, CXCR3 and CX3CR1) and other markers of cell activation (MHC class I and II, CR3) was determined at the mRNA level by semiquantitative RT-PCR and at the protein level by confocal microscopy. Rat peritoneal macrophages were loaded with a fluorescent cell-tracer and their ability to infiltrate inflamed aggregates treated with IFNγ was analysed by FACS and confocal microscopy.

Liver failure, whether arising directly from acute liver failure or from decompensated chronic liver disease is an increasing problem worldwide and results in many deaths. In the UK only 10% of individuals requiring a liver transplant receive one. Thus the need for alternative treatments is paramount. A BioArtificial Liver machine could temporarily replace the functions of the liver, buying time for the patient's liver to repair and regenerate. We have designed, implemented and tested a clinical-scale BioArtificial Liver machine containing a biomass derived from a hepatoblastoma cell-line cultured as three dimensional organoids, using a fluidised bed bioreactor, together with single-use bioprocessing equipment, with complete control of nutrient provision with feedback BioXpert recipe processes, and yielding good phenotypic liver functions. The methodology has been designed to meet specifications for GMP production, required for manufacture of advanced therapy medicinal products (ATMPs). In a porcine model of severe liver failure, damage was assured in all animals by surgical ischaemia in pigs with human sized livers (1.2–1.6 kg liver weights). The BioArtificial liver (UCLBAL) improved important prognostic clinical liver-related parameters, eg, a significant improvement in coagulation, reduction in vasopressor requirements, improvement in blood pH and in parameters of intracranial pressure (ICP) and oxygenation. Acute liver failure (ALF) is frequently a catastrophic event as the clinical course is often complicated by multi-organ failure with cerebral oedema being the terminal event. Acute liver failure can occur at any age, is commonly idiosyncratic with no specific therapy and patients present to hospital critically ill. Approximately 15% will recover spontaneously; the remainder fulfil the Kings College transplant criteria and 85% of those are listed on an urgent transplant waiting list. Currently in UK more than 20% of patients die on the waiting list. Liver disease is the only major disease in the UK on the increase, whilst donor organ availability is almost static, thus there is a large unmet medical need for an alternative treatment. Since the liver has the capacity to repair and regenerate given time, a solution is to provide temporary liver function to " buy time " either for complete recovery or to find a suitable donor organ. We have developed a bio-artificial liver machine based on a biomass comprised of human-liver derived cells 1–4 combined with adsorptive removal of DNA and endotoxin, to be used in an extracor-poreal circuit, treating the whole plasma fraction of the patient over several hours. The human liver is 1.2–1.6 kg containing 1–2 × 10 11 hepatocytes; experimental and clinical evidence demonstrates that patients can survive even if they have only ~30% of liver function 5. The aim of this study was to develop methodology appropriate to GMP production for a cell therapy to be delivered on the clinical scale, delivering ~70 billion cells and to test it in a severe, non-reversible model of acute liver failure in pigs with liver weights equivalent to those in humans.

Biomaterials prepared from polyesters of lactic acid and glycolic acid, or a mixture of the two, degrade in the presence of water into the naturally occurring metabolites, lactic acid and glycolic acid. While the lactic acid degradation product that is released from biomaterials is well tolerated by the body, lactic acid can influence the metabolic function of cells; it can serve as an energy substrate for cells, and has been shown to have antioxidant properties. Neural precursor cells, a cell population of considerable interest as a source of cells for neural tissue regeneration strategies, generate a high amount of reactive oxygen species, and when associated with a degradable biomaterial, may be impacted by released lactic acid. In this work, the effect of lactic acid on a neural cell population containing proliferative neural precursor cells was examined in monolayer culture. Lactic acid was found to scavenge exogenously added free radicals produced in the presence of either hydrogen peroxide or a photoinitiator (I2959) commonly utilized in the preparation of photopolymerizable biomaterials. In addition to its effect on exogenously added free radicals, lactic acid reduced intracellular redox state, increased the proliferation of the cell population, and modified the cell composition. The findings of this study provide insight into the role that lactic acid plays naturally on developing neural cells and are also of interest to biomaterials scientists that are focused on the development of degradable lactic-acid-based polymers for cell culture devices. The effect of lactic acid on other cell populations may differ and should be characterized to best understand how cells function in degradable cell culture devices.

Tumor-originated and undefined extracel-lular matrices (ECMs) such as Matrigel TM have been widely used in three-dimensional (3D) cell and tissue culture, but their use is unacceptable in clinical cell therapies. In this study, we proposed a 3D cellulose nanofiber (CNF) hydrogel that has great potential as a defined tissue-engineering scaffold, especially for osteoblast culture. The CNF hydrogel showed attractive features as a cell scaffold material. It exhibited a * 1.4-fold higher diffusion coefficient (* 2.98 9 10-7 cm 2 / s) of macromolecules such as bovine serum albumin than does Matrigel TM (\ 2.2 9 10-7 cm 2 /s) due to the former's higher porosity ([ 95%) and pore size (* 310.8 lm). Most pre-osteoblast cells that are encapsulated in the CNF hydrogel were immediately locked without sinking by instant hydrogen bond cross-linking between CNFs, whereas cells encapsulated in Matrigel TM sank to the bottom of the scaffold due to the slow sol-gel transition ([ 20 min). The elastic modulus of the cell-encapsulated CNF hydrogel could be reinforced by further calcium-mediated cross-linking without cytotoxicity. As a result, the pre-osteoblast cells in the CNF hydrogels were homogeneously distributed in the 3D structure, proliferated for 3 weeks, and successfully differentiated. Overall, CNFs showed that it has potential to be used in tissue engineering as a defined ECM component. Hyo Jeong Kim and Dongyeop X. Oh have contributed equally to this work and are considered first co-authors.

Mesenchymal stem/stromal cells (MSCs) are considered an important candidate in cell therapy and tissue engineering approaches. The culture of stem cells in a 3D environment is known to better resemble the in vivo situation and to promote therapeutically relevant effects in isolated cells. Therefore, the aim of this study was to develop an approach for the isolation of MSCs from adipose tissue into a 3D environment. Furthermore, the use of cryoprotective medium for cryopreservation of whole adipose tissue was evaluated. For the isolation of MSCs, a novel human platelet lysate-based hydrogel was used as matrix and the migration, yield, viability and metabolic activity of cells from the 3D matrix were compared to cells from 2D explant culture. Also, the surface marker profile and differentiation capacity of MSCs from the 3D matrix were evaluated and compared to MSCs from isolation by enzymatic treatment. We found that cryopreservation of whole adipose tissue is feasible, and therefore adipose tissue can be stored and is available for MSC isolation on demand. Also, we demonstrate the isolation of MSCs into the 3D matrix and that cells from this condition display a similar phenotype and differentiation capacity like MSCs derived by traditional isolation procedure.  The presented approach allows, for the first time, to isolate MSCs directly into a soft 3D hydrogel environment, avoiding any contact to a 2D plastic culture surface.

Mesenchymal stem cells (MSCs) are considered as primary candidates for cell-based therapies due to their multiple effects in regenerative medicine. Pre-conditioning of MSCs under physiological conditions—such as hypoxia, three-dimensional environments, and dynamic cultivation—prior to transplantation proved to optimize their therapeutic efficiency. When cultivated as three-dimensional aggregates or spheroids, MSCs display increased angiogenic, anti-inflammatory, and immunomodulatory effects as well as improved stemness and survival rates after transplantation, and cultivation under dynamic conditions can increase their viability, proliferation, and paracrine effects, alike. Only few studies reported to date, however, have utilized dynamic conditions for three-dimensional aggregate cultivation of MSCs. Still, the integration of dynamic bioreactor systems, such as spinner flasks or stirred tank reactors might pave the way for a robust, scalable bulk expansion of MSC aggregates or MSC-derived extracellular vesicles. This review summarizes recent insights into the therapeutic potential of MSC aggregate cultivation and focuses on dynamic generation and cultivation techniques of MSC aggregates.

We introduce an agent-based model of epithelial cell morphogenesis to explore the complex interplay between apoptosis, proliferation, and polarization. By varying the activity levels of these mechanisms we derived phenotypic transition maps of normal and aberrant morphogenesis. These maps identify homeostatic ranges and morphologic stability conditions. The agent-based model was parameterized and validated using novel high-content image analysis of mammary acini morphogenesis in vitro with focus on time-dependent cell densities, proliferation and death rates, as well as acini morphologies. Model simulations reveal apoptosis being necessary and sufficient for initiating lumen formation, but cell polarization being the pivotal mechanism for maintaining physiological epithelium morphology and acini sphericity. Furthermore, simulations highlight that acinus growth arrest in normal acini can be achieved by controlling the fraction of proliferating cells. Interestingly, our simulations reveal a synergism between polarization and apoptosis in enhancing growth arrest. After validating the model with experimental data from a normal human breast line (MCF10A), the system was challenged to predict the growth of MCF10A where AKT-1 was overexpressed, leading to reduced apoptosis. As previously reported, this led to non growth-arrested acini, with very large sizes and partially filled lumen. However, surprisingly, image analysis revealed a much lower nuclear density than observed for normal acini. The growth kinetics indicates that these acini grew faster than the cells comprising it. The in silico model could not replicate this behavior, contradicting the classic paradigm that ductal carcinoma in situ is only the result of high proliferation and low apoptosis. Our simulations suggest that overexpression of AKT-1 must also perturb cell–cell and cell–ECM communication, reminding us that extracellular context can dictate cellular behavior.

This study aims at identifying compositional and architectural (pore size
and distribution) parameters of biocompatible scaffolds, which can be best suitable for both osteoblasts and endothelial cells to produce optimized 3D cocultured constructs. Spongy scaffolds are prepared using poly(vinyl alcohol) (PVA) and gelatin (G) at different weight compositions (PVA/G range:100/0–50/50, w/w) via emulsion and freeze-drying. The higher the gelatin content, the larger is the volume occupied by higher size pores. Human umbilical vein endothelial cells and human mesenchymal stromal cells are independently differentiated on the scaffolds to select the best candidate for the co-culture. The results of metabolic activity and histology on single platforms show both cell- and material-type dependent outcomes. PVA/G 80/20 scaffolds are fially selected and allow the formation of mineralized matrix containing organized endothelial-like structures. This study highlights the need for systematic investigations on multifactorial parameters of scaffolds to
improve vascularized bone substitutes.

Background: Mesenchymal stem/stromal cells (MSCs) are considered an important candidate in cell therapy and tissue engineering approaches. The culture of stem cells in a 3D environment is known to better resemble the in vivo situation and to promote therapeutically relevant effects in isolated cells. Therefore, the aim of this study was to develop an approach for the direct isolation of MSCs from adipose tissue into a 3D environment, avoiding contact to a 2D plastic surface. Furthermore, the use of a cryoprotective medium for the cryopreservation of whole adipose tissue was evaluated. Materials and methods: Cryopreservation of fresh adipose tissue with and without a cryoprotective medium was compared with regard to the viability and metabolic activity of cells. After thawing, the tissue was embedded in a novel human platelet lysate-based hydrogel for the isolation of MSCs. The migration, yield, viability, and metabolic activity of cells from the 3D matrix were compared to cells from 2D explant culture. Also, the surface marker profile and differentiation capacity of MSCs from the 3D matrix were evaluated and compared to MSCs from isolation by enzymatic treatment or 2D explant culture.

LAM is a rare low-grade metastasizing lung neoplasm. Inhibitors of mTOR improve clinical outcome of
LAM patients by preventing loss of lung function. Nevertheless, other cell targets may be of interest for
drug development. Therefore, we explored the potential role of EDN1 (endothelin) in LAM. We report
an increased endothelin blood level in LAM patients as well as EDN1 overexpression and EDN1 receptor
downregulation in LAM-derived primary cells and in TSC2NEG cells mutated in TSC2. We evidenced EDN
pathway dysregulation based on EDN1, EDNRA, EDNRB and ARRB1 mRNA expression in LAM-derived
primary cells. We showed overexpression of EDN1 and ARRB1 mRNAs in TSC2NEG cells; these cells lost
their ability to respond to stimulation by endothelin. We analyzed the effects of endothelin receptor
antagonists alone or in combination with rapamycin, an mTOR inhibitor, on proliferation and migration
of LAM cells. Rapamycin treatment of TSC2NEG cells significantly reduced cell proliferation or migration,
while none of the tested inhibitors of EDN receptors impaired these functions. We showed that
TSC2NEG cells have acquired a transformed phenotype as showed by their ability to grow as spheroids in
semi-solid medium and that unlike endothelin receptors antagonists, rapamycin reduced anchorageindependent
cell growth and prevented expansion of TSC2NEG spheroids.

We are taking an in vitro approach to study the effect of inflammatory mediators on cells in the rat central nervous system (CNS) and to assess their contribution (particularly microglia and astrocytes) to the inflammatory response. To this end, a three-dimensional aggregate culture system derived from foetal rat brain tissue which approximates to the brain in vivo in terms of glial and neuronal development and cell–cell interactions is used as a model of the CNS parenchyma.
Aggregate cultures are established by seeding dissociated foetal rat brain cells (Sprague–Dawley embryos at 16-days’ gestation) over agar to prevent adherence. Under these conditions, the cells form floating aggregates, which can be cultured for up to 6 weeks. Proliferating cells, identified by Bromodeoxyuridine (BrdU) incorporation and double labelling, include GFAP+ astrocytes. Basal levels of MHC classes I and II and CR3 expression are very low, as determined by immunocytochemistry and confocal microscopy. Treatment of aggregates with IFN-gamma (48 h, 50 ng/ml) results in increased MHC class I and class II expression. Preliminary results will be presented to illustrate how aggregate culture of foetal brain cells can be used to assess the effect of inflammatory mediators on cells within a complex CNS environment, complementing existing studies on isolated cell populations and co-cultures.

Respiratory viral infections, including severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2), are among the most common illnesses and a leading cause of morbidity and mortality
worldwide. Due to the severe effects on health, the need of new tools to study the pathogenesis of
respiratory viruses as well as to test for new antiviral drugs and vaccines is urgent. In vitro culture
model systems, such as three-dimensional (3D) cultures, are emerging as a desirable approach to
understand the virus host interactions and to identify novel therapeutic agents. In the first part of the
article, we address the various scaffold-free and scaffold-based 3D culture models such as hydrogels,
bioreactors, spheroids and 3D bioprinting as well as present their properties and advantages over
conventional 2D methods. Then, we review the 3D models that have been used to study the most
common respiratory viruses including influenza, parainfluenza, respiratory syncytial virus (RSV)
and coronaviruses. Herein, we also explain how 3D models have been applied to understand the
novel SARS-CoV-2 infectivity and to develop potential therapies.

As life expectancy grows, many age-related diseases are becoming increasingly evident such as osteoporosis, diabetes or cardiovascular disorders. Not only that, some studies are also pointing up an increase of road accidents and trauma injuries boosting the implants market. Many techniques have been developed in order to repair damaged organs, and Tissue Engineering (TE) is a promising one. In order to reduce the dependence on transplanted tissues and organs, TE combines the field of life science with engineering by developing a 3D template (scaffold) which temporarily mimics an extracellular matrix (ECM) and provides a support for cells to grow and proliferate with the right stimuli (signals). Not only may the source of the cells and signals vary, but also the scaffold itself. Depending on the purpose, there are many possibilities in which the scaffold can be designed: ceramic materials, synthetic or natural polymers with a vast range of architecture. Creating a system as similar as possible to ECM is advantageous for the cells, for this reason fibrous structures and natural polymers, such as collagen and gelatin, are outstanding regarding TE.

Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events during angio-genesis in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by undefined substrates and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). We rapidly encapsulated iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydro-gel spheres using thiol-ene chemistry and subsequently encapsulated cell-dense hydrogel spheres in a cell-free hydrogel layer. The hydrogel sprouting array supported pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. iPSC-ECs in the sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors of vascular endothelial growth factor, NF-jB, matrix metalloproteinase-2/9, protein kinase activity, and b-tubulin, which confirms their functional role in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds from the US Environmental Protection Agency's ToxCast library identified six compounds that inhibited iPSC-EC sprouting and five compounds that were overtly cytotoxic to iPSC-ECs at a single concentration. The chemically-defined iPSC-EC sprouting model (iSM) is thus amenable to enhanced-throughput screening of small molecular libraries for effects on angiogenic sprouting and iPSC-EC toxicity assessment. Statement of Significance Angiogenesis assays that are commonly used for drug screening and toxicity assessment applications typically utilize natural substrates like Matrigel TM that are difficult to spatially pattern, costly, ill-defined, and may exhibit lot-to-lot variability. Herein, we describe a novel angiogenic sprouting assay using chemically-defined, bioinert poly(ethylene glycol) hydrogels functionalized with biomimetic peptides to promote cell attachment and degradation in a reproducible format that may mitigate the need for natural substrates. The quantitative assay of angiogenic sprouting here enables precise control over the initial conditions and can be formulated into arrays for screening. The sprouting assay here was dependent on key angiogenic signaling axes in a screen of angiogenesis inhibitors and a blinded screen of putative vascular disrupting compounds from the US-EPA.