Cell Culture Tools Research Articles

Advancing Synthetic Hydrogels through Nature-Inspired Materials Chemistry.

Synthetic extracellular matrix (ECM) mimics that can recapitulate the complex biochemical and mechanical nature of native tissues are needed for advanced models of development and disease. Biomedical research has heavily relied on the use of animal-derived biomaterials, which is now impeding their translational potential and convoluting the biological insights gleaned from in vitro tissue models. Natural hydrogels have long served as a convenient and effective cell culture tool, but advances in materials chemistry and fabrication techniques now present promising new avenues for creating xenogenic-free ECM substitutes appropriate for organotypic models and microphysiological systems. However, significant challenges remain in creating synthetic matrices that can approximate the structural sophistication, biochemical complexity, and dynamic functionality of native tissues. This review summarizes key properties of the native ECM, and discusses recent approaches used to systematically decouple and tune these properties in synthetic matrices. The importance of dynamic ECM mechanics, such as viscoelasticity and matrix plasticity, is also discussed, particularly within the context of organoid and engineered tissue matrices. Emerging design strategies to mimic these dynamic mechanical properties are reviewed, such as multi-network hydrogels, supramolecular chemistry, and hydrogels assembled from biological monomers.

Abstract A078: Characterization of normal human male mammary cells and their de novo transformed derivatives

Abstract Men normally develop and maintain bi-layered mammary glands that appear structurally and histologically similar to those that develop in women, but with reported deficient lobule formation. Men also develop breast cancer, mostly of the ER+ subtype and an age-associated increasing onset, but at a 100-fold lower incidence and overall worse outcome. However, the classification and treatment of male breast cancers are largely based on strategies developed for female patients, despite their known hormonal and other differences. This is likely due, at least in part, to the fact that adult male mice, unlike humans do not develop full mammary glands. Thus, experimental access to mouse models have been lacking and studies of “normal” male mammary tissue have thus been largely restricted to analysis of human male gynecomastia samples. We now report the utility of multiple systems for analyzing normal adult human female mammary cell properties to this source of freshly obtained and viably cryopreserved normal human male mammary tissue. Histological examination of male mammary tissues (3 donors) confirmed their reported bi-layered structure surrounded by fibroblasts and lack of gross evidence of lobules. Use of procedures created for analyzing female breast tissue indicates that the human male mammary glands contain the same subsets but in different relative proportions; i.e., the proportion of EpCAM+CD49f- mammary cells (Luminal Cells, LCs) is relatively increased and of the EpCAM+CD49f+ cells (Luminal Progenitors, LPs) and EpCAM-CD49f+ cells (Basal Cells, BCs) is correspondingly decreased, consistent with a lack of alveolae. However, the freshly isolated male LPs and BCs contain a similar frequency of EGF-responsive colony-forming cells (CFCs) as female LPs and BCs. Subcutaneous transplantation of the male mammary cells in both male and female immunodeficient mice resulted in the generation of hollow mammary structures 4 weeks later as previously shown for female cells, and these were also found to contain LC, LP and BC populations including some with detectable levels of CFCs suggesting their derivation from a male mammary stem cell population (Eirew et al Nat Med 2008). We have also found evidence from qPCR and RNA-seq analyses of reduced progesterone signaling and alveologenesis in male LPs and LCs. These findings suggest hormonal differences may play a major role in regulating the altered ratios of cell types present in the normal adult human male mammary gland in comparison to premenopausal female. Interestingly, preliminary experiments also indicate that normal human male mammary cells transduced with KRASG12D without, or with BMI1+MYC+TP53R273C , generate analogous slowly-growing ER+, and rapidly-growing, highly aggressive tumours, respectively, in male immunodeficient mice. Taken together, our findings demonstrate the potential of established cell culture, xenotransplantation and molecular analytical tools to reveal the altered mechanisms that account for the different structure and biology of human male mammary tissue. Citation Format: Shengsen Ding, Susanna Tan, Ebrahim Eskandari-Nasab, Shrinka Sen, Martin Hirst, Connie Eaves. Characterization of normal human male mammary cells and their de novo transformed derivatives [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A078.

Electroactive 4D Porous Scaffold Based on Conducting Polymer as a Responsive and Dynamic In Vitro Cell Culture Platform.

In vivo, cells reside in a 3D porous and dynamic microenvironment. It provides biochemical and biophysical cues that regulate cell behavior in physiological and pathological processes. In the context of fundamental cell biology research, tissue engineering, and cell-based drug screening systems, a challenge is to develop relevant in vitro models that could integrate the dynamic properties of the cell microenvironment. Taking advantage of the promising high internal phase emulsion templating, we here designed a polyHIPE scaffold with a wide interconnected porosity and functionalized its internal 3D surface with a thin layer of electroactive conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) to turn it into a 4D electroresponsive scaffold. The resulting scaffold was cytocompatible with fibroblasts, supported cellular infiltration, and hosted cells, which display a 3D spreading morphology. It demonstrated robust actuation in ion- and protein-rich complex culture media, and its electroresponsiveness was not altered by fibroblast colonization. Thanks to customized electrochemical stimulation setups, the electromechanical response of the polyHIPE/PEDOT scaffolds was characterized in situ under a confocal microscope and showed 10% reversible volume variations. Finally, the setups were used to monitor in real time and in situ fibroblasts cultured into the polyHIPE/PEDOT scaffold during several cycles of electromechanical stimuli. Thus, we demonstrated the proof of concept of this tunable scaffold as a tool for future 4D cell culture and mechanobiology studies.

WITHDRAWN: In Vitro three-dimensional (3D) cell culture tools for spheroid and organoid models

Three-dimensional (3D) cell culture technology has been steadily studied since the 1990′s due to its superior biocompatibility compared to the conventional two-dimensional (2D) cell culture technology, and has recently developed into an organoid culture technology that further improved biocompatibility. Since the 3D culture of human cell lines in artificial scaffolds was demonstrated in the early 90′s, 3D cell culture technology has been actively developed owing to various needs in the areas of disease research, precision medicine, new drug development, and some of these technologies have been commercialized. In particular, 3D cell culture technology is actively being applied and utilized in drug development and cancer-related precision medicine research. Drug development is a long and expensive process that involves multiple steps—from target identification to lead discovery and optimization, preclinical studies, and clinical trials for approval for clinical use. Cancer ranks first among life-threatening diseases owing to intra-tumoral heterogeneity associated with metastasis, recurrence, and treatment resistance, ultimately contributing to treatment failure and adverse prognoses. Therefore, there is an urgent need for the development of efficient drugs using 3D cell culture techniques that can closely mimic in vivo cellular environments and customized tumor models that faithfully represent the tumor heterogeneity of individual patients. This review discusses 3D cell culture technology focusing on research trends, commercialization status, and expected effects developed until recently. We aim to summarize the great potential of 3D cell culture technology and contribute to expanding the base of this technology.

Critical appraisal tools used in systematic reviews of in vitro cell culture studies: A methodological study.

Systematic reviews (SRs) of preclinical studies are marked with poor methodological quality. In vitro studies lack assessment tools to improve the quality of preclinical research. This methodological study aimed to identify, collect, and analyze SRs based on cell culture studies to highlight the current appraisal tools utilized to support the development of a validated critical appraisal tool for cell culture in vitro research. SRs, scoping reviews, and meta-analyses that included cell culture studies and used any type of critical appraisal tool were included. Electronic search, study selection, data collection and methodological quality (MQ) assessment tool were realized. Further, statistical analyses regarding possible associations and correlations between MQ and collected data were performed. After the screening process, 82 studies remained for subsequent analysis. A total of 32 different appraisal tools were identified. Approximately 60% of studies adopted pre-structured tools not designed for cell culture studies. The most frequent instruments were SYRCLE (n = 14), OHAT (n = 9), Cochrane Collaboration's tool (n = 7), GRADE (n = 6), CONSORT (n = 5), and ToxRTool (n = 5). The studies were divided into subgroups to perform statistical analyses. A significant association (OR = 5.00, 95% CI = 1.54-16.20, p = 0.008) was found between low MQ and chronic degenerative disorders as topic of SR. Several challenges in collecting information from the included studies led to some modifications related to the previously registered protocol. These results may serve as a basis for further development of a critical appraisal tool for cell culture studies capable of capturing all the essential factors related to preclinical research, therefore enhancing the practice of evidence-based.

A novel study of melatonin diffusion in a 3D cell culture model

Melatonin is now considered a major physiological regulator of many different functions including synchronization of circadian rhythms, antioxidant defense at different levels, immunomodulation, cell growth control, neuroprotector and anti-tumor agent. In addition to its membrane receptor-dependent actions, it has been classically assumed that its diffusion through lipid bilayers contribute to its intracellular actions, including direct and indirect free radical scavenging activities. While pineal gland is the major site of nocturnal production of the indolamine, skin is considered an important source of melatonin synthesis. Here, using a 3-D culture model of HaCaT cells in an artificial scaffold (epidermal equivalents), we have quantified diffusion of melatonin in these cells and compared it to 2-D or spheroid cultures. Diffusion in 3-D scaffold cultures was similar to that found in 2-D culture and proportion of intracellular melatonin was low. AFMK, a major oxidative metabolite of melatonin, was also found and quantified. Redox parameters including total ROS, superoxide or mitochondrial mass were also assayed. We also report the effect of melatonin on the cytoskeleton of normal human keratinocyte HaCaT cells. We propose HaCaT epidermal equivalents as an affordable, easy-to-use, 3-D cell culture tool to test diffusion rates of melatonin but also other similar small molecules. This 3-D models can also be studied at cellular and molecular level, including redox parameters, and can provide important information regarding molecules that can be topically added to skin. Similarly, mechanisms of transportation can also be approached with this methodology.

In Vitro three-dimensional (3D) cell culture tools for spheroid and organoid models.

Three-dimensional (3D) cell culture technology has been steadily studied since the 1990's due to its superior biocompatibility compared to the conventional two-dimensional (2D) cell culture technology, and has recently developed into an organoid culture technology that further improved biocompatibility. Since the 3D culture of human cell lines in artificial scaffolds was demonstrated in the early 90's, 3D cell culture technology has been actively developed owing to various needs in the areas of disease research, precision medicine, new drug development, and some of these technologies have been commercialized. In particular, 3D cell culture technology is actively being applied and utilized in drug development and cancer-related precision medicine research. Drug development is a long and expensive process that involves multiple steps-from target identification to lead discovery and optimization, preclinical studies, and clinical trials for approval for clinical use. Cancer ranks first among life-threatening diseases owing to intra-tumoral heterogeneity associated with metastasis, recurrence, and treatment resistance, ultimately contributing to treatment failure and adverse prognoses. Therefore, there is an urgent need for the development of efficient drugs using 3D cell culture techniques that can closely mimic in vivo cellular environments and customized tumor models that faithfully represent the tumor heterogeneity of individual patients. This review discusses 3D cell culture technology focusing on research trends, commercialization status, and expected effects developed until recently. We aim to summarize the great potential of 3D cell culture technology and contribute to expanding the base of this technology.

A review of open-source image analysis tools for mammalian cell culture: algorithms, features and implementations.

Cell culture is undeniably important for multiple scientific applications, including pharmaceuticals, transplants, and cosmetics. However, cell culture involves multiple manual steps, such as regularly analyzing cell images for their health and morphology. Computer scientists have developed algorithms to automate cell imaging analysis, but they are not widely adopted by biologists, especially those lacking an interactive platform. To address the issue, we compile and review existing open-source cell image processing tools that provide interactive interfaces for management and prediction tasks. We highlight the prediction tools that can detect, segment, and track different mammalian cell morphologies across various image modalities and present a comparison of algorithms and unique features of these tools, whether they work locally or in the cloud. This would guide non-experts to determine which is best suited for their purposes and, developers to acknowledge what is worth further expansion. In addition, we provide a general discussion on potential implementations of the tools for a more extensive scope, which guides the reader to not restrict them to prediction tasks only. Finally, we conclude the article by stating new considerations for the development of interactive cell imaging tools and suggesting new directions for future research.

Traumatic Brain Injury in a Well: A Modular Three-Dimensional Printed Tool for Inducing Traumatic Brain Injury In vitro.

Traumatic brain injury (TBI) is a major health problem that affects millions of persons worldwide every year among all age groups, mainly young children, and elderly persons. It is the leading cause of death for children under the age of 16 and is highly correlated with a variety of neuronal disorders, such as epilepsy, and neurodegenerative disease, such as Alzheimer's disease or amyotrophic lateral sclerosis. Over the past few decades, our comprehension of the molecular pathway of TBI has improved, yet despite being a major public health issue, there is currently no U.S. Food and Drug Administration-approved treatment for TBI, and a gap remains between these advances and their application to the clinical treatment of TBI. One of the major hurdles for pushing TBI research forward is the accessibility of TBI models and tools. Most of the TBI models require costume-made, complex, and expensive equipment, which often requires special knowledge to operate. In this study, we present a modular, three-dimensional printed TBI induction device, which induces, by the pulse of a pressure shock, a TBI-like injury on any standard cell-culture tool. Moreover, we demonstrate that our device can be used on multiple systems and cell types and can induce repetitive TBIs, which is very common in clinical TBI. Further, we demonstrate that our platform can recapitulate the hallmarks of TBI, which include cell death, decrease in neuronal functionality, axonal swelling (for neurons), and increase permeability (for endothelium). In addition, in view of the continued discussion on the need, benefits, and ethics of the use of animals in scientific research, this in vitro, high-throughput platform will make TBI research more accessible to other labs that prefer to avoid the use of animals yet are interested in this field. We believe that this will enable us to push the field forward and facilitate/accelerate the availability of novel treatments.

Thyroid-Friendly Soft Materials as 3D Cell Culture Tool for Stimulating Thyroid Cell Function.

The disruption of thyroid hormones because of chemical exposure is a significant societal problem. Chemical evaluations of environmental and human health risks are conventionally based on animal experiments. However, owing to recent breakthroughs in biotechnology, the potential toxicity of chemicals can now be evaluated using 3D cell cultures. In this study, the interactive effects of thyroid-friendly soft (TS) microspheres on thyroid cell aggregates are elucidated and their potential as a reliable toxicity assessment tool is evaluated. Using state-of-the-art characterization methods coupled with cell-based analysis and quadrupole time-of-flight mass spectrometry, it is shown that TS-microsphere-integrated thyroid cell aggregates exhibit improved thyroid function. Specifically, the responses of zebrafish embryos, which are used for thyroid toxicity analysis, and the TS-microsphere-integrated cell aggregates to methimazole (MMI), a known thyroid inhibitor, are compared. The results show that the thyroid hormone disruption response of the TS-microsphere-integrated thyroid cell aggregates to MMI is more sensitive compared with those of the zebrafish embryos and conventionally formed cell aggregates. This proof-of-concept approach can be used to control cellular function in the desired direction and hence evaluate thyroid function. Thus, the proposed TS-microsphere-integrated cell aggregates may yield new fundamental insights for advancing in vitro cell-based research.

Gravity-driven microfluidic device placed on a slow-tilting table enables constant unidirectional perfusion culture of human induced pluripotent stem cells

Gravity-driven microfluidics, which utilizes gravity force to drive liquid flow, offers portability and multi-condition setting flexibility because they do not require pumps or connection tubes to drive the flow. However, because the flow rate decreases with time in gravity-driven microfluidics, it is not suitable for stem cell experiments, which require long-term (at least a day) stability. In this study, gravity-driven microfluidics and a slow-tilting table were developed to culture cells under constant unidirectional perfusion. The microfluidic device was placed on a slow-tilting table, which tilts unidirectionally at a rate of approximately 7° per day to compensate for the reduction in the flow rate. Computational simulations showed that the pulsation of the flow arising from the stepwise movement of the table was less than 0.2%, and the flow was laminar. Hydrophilization of the tanks increased the flow rate, which is consistent with the theoretical values. We showed that vitronectin is better than laminin 511 fragments as a coating material for adhering human induced pluripotent stem cells on a microchamber made of polydimethylsiloxane, and succeeded in culturing the cells for 3 days. It is believed that the system offers easy-to-use cell culture tools, such as conventional multiwell culture vessels, and enables the control of the cell microenvironment.

From Tissue Physoxia to Cancer Hypoxia, Cost-Effective Methods to Study Tissue-Specific O2 Levels in Cellular Biology.

The human body is endowed with an extraordinary ability to maintain different oxygen levels in various tissues and organs. The maintenance of physiological levels of oxygen is known as physoxia. The development of hypoxic conditions plays an important role in the biology of several pathologies, including cancer. In vitro studies using normal and neoplastic cells require that culture conditions be carried out under appropriate oxygen levels, either physoxic or hypoxic conditions. Such requirements are difficult to widely implement in laboratory practice, mainly due to the high costs of specialized equipment. In this work, we present and characterize a cost-effective method to culture cells under a range of oxygen levels using deoxidizing pouches. Our results show that physoxic and hypoxic levels using deoxidizing absorbers can be achieved either by implementing a gradual change in oxygen levels or by a regimen of acute depletion of oxygen. This approach triggers the activation of an epithelial-mesenchymal transition in cancer cells while stimulating the expression of HIF-1α. Culturing cancer cells with deoxidizing agent pouches revealed PI3K oncogenic pathway exacerbations compared to tumor cells growing under atmospheric levels of oxygen. Similar to the PI3K signaling disturbance, we also observed augmented oxidative stress and superoxide levels and increased cell cycle arrest. Most interestingly, the culture of cancer cells under hypoxia resulted in the accumulation of cancer stem cells in a time-dependent manner. Overall, we present an attractive, cost-effective method of culturing cells under appropriate physoxic or hypoxic conditions that is easily implementable in any wet laboratory equipped with cell culture tools.

Applications of Digital Health in Public Health: The China Experience

Applications of Digital Health in Public Health: The China Experience

Organoid transplant approaches for the liver.

Organoid technology is a state-of-the-art cell culture tool that has revolutionized study of development, regeneration, and diseases. Human liver organoids (HLOs) are now derived from either adult stem/progenitors or pluripotent stem cells (PSCs), emulating cellular diversity and structural symphony akin to the human liver. With the rapid rise in decompensated liver disease conditions only treated by liver transplant therapy, HLOs represent an alternate source for transplantation to address the ongoing shortage of grafts. Although ongoing advancements in bioengineering technology have moved the organoid transplant approach to the next level, sustained survival of the transplanted tissue still eludes us toward functional organ replacement. Herein, we review the development of HLOs and discuss promises and challenges on organoid transplant approaches.

Growth and eGFP Production of CHO-K1 Suspension Cells Cultivated From Single Cell to Laboratory Scale.

Scaling down bioproduction processes has become a major driving force for more accelerated and efficient process development over the last decades. Especially expensive and time-consuming processes like the production of biopharmaceuticals with mammalian cell lines benefit clearly from miniaturization, due to higher parallelization and increased insights while at the same time decreasing experimental time and costs. Lately, novel microfluidic methods have been developed, especially microfluidic single-cell cultivation (MSCC) devices have been proved to be valuable to miniaturize the cultivation of mammalian cells. So far, growth characteristics of microfluidic cultivated cell lines were not systematically compared to larger cultivation scales; however, validation of a miniaturization tool against initial cultivation scales is mandatory to prove its applicability for bioprocess development. Here, we systematically investigate growth, morphology, and eGFP production of CHO-K1 cells in different cultivation scales ranging from a microfluidic chip (230 nl) to a shake flask (125 ml) and laboratory-scale stirred tank bioreactor (2.0 L). Our study shows a high comparability regarding specific growth rates, cellular diameters, and eGFP production, which proves the feasibility of MSCC as a miniaturized cultivation tool for mammalian cell culture. In addition, we demonstrate that MSCC provides insights into cellular heterogeneity and single-cell dynamics concerning growth and production behavior which, when occurring in bioproduction processes, might severely affect process robustness.

A new cell-sized support for 3D cell cultures based on recombinant spider silk fibers.

It is now generally accepted that 2D cultures cannot accurately replicate the rich environment and complex tissue architecture that exists in vivo, and that classically cultured cells tend to lose their original function. Growth of spheroids as opposed to 2D cultures on plastic has now been hailed as an efficient method to produce quantities of high-quality cells for cancer research, drug discovery, neuroscience, and regenerative medicine. We have developed a new recombinant protein that mimics dragline spidersilk and that self-assembles into cell-sized coils. These have high thermal and shelf-life stability and can be readily sterilized and stored for an extended period of time. The fibers are flexible, elastic, and biocompatible and can serve as cell-sized scaffold for the formation of 3D cell spheroids. As a proof of concept, recombinant spidersilk was integrated as a scaffold in spheroids of three cell types: primary rat hepatocytes, human mesenchymal stem cells, and mouse L929 cells. The scaffolds significantly reduced spheroid shrinkage and unlike scaffold-free spheroids, spheroids did not disintegrate over the course of long-term culture. Cells in recombinant spidersilk spheroids showed increased viability, and the cell lines continued to proliferate for longer than control cultures without spidersilk. The spidersilk also supported biological functions. Recombinant spidersilk primary hepatocyte spheroids exhibited 2.7-fold higher levels of adenosine triphosphate (ATP) continued to express and secrete albumin and exhibited significantly higher basal and induced CYP3A activity for at least 6 weeks in culture, while control spheroids without fibers stopped producing albumin after 27 days and CPY3A activity was barely detectable after 44 days. These results indicate that recombinant spidersilk can serve as a useful tool for long-term cell culture of 3D cell spheroids and specifically that primary hepatocytes can remain active in culture for an extended period of time which could be of great use in toxicology testing.

An Improved Scalable Hydrogel Dish for Spheroid Culture.

Research in fields studying cellular response to surface tension and mechanical forces necessitate cell culture tools with tunability of substrate stiffness. We created a scalable hydrogel dish design to facilitate scaffold-free formation of multiple spheroids in a single dish. Our novel design features inner and outer walls, allowing efficient media changes and downstream experiments. The design is easily scalable, accommodating varying numbers of microwells per plate. We report that non-adherent hydrogel stiffness affects spheroid morphology and compaction. We found that spheroid morphology and viability in our hydrogel dishes were comparable to commercially available Aggrewell™800 plates, with improved tunability of surface stiffness and imaging area. Device function was demonstrated with a migration assay using two investigational inhibitors against EMT. We successfully maintained primary-derived spheroids from murine and porcine lungs in the hydrogel dish. These features increase the ability to produce highly consistent cell aggregates for biological research.

Cross talk between hepatitis B virus and innate immunity of hepatocytes.

Innate immunity plays a major role in controlling viral infections. Recent exploration of sodium taurocholate co-transporting polypeptide receptor as specific hepatitis B virus (HBV) receptor in human hepatocytes has provided appropriate cell culture tools to study the innate immunity of hepatocytes and its cross talk with HBV. In this review, we give a brief update on interaction between HBV and innate immunity using the currently available in vitro cellular models that support the complete life cycle of HBV. We will discuss how HBV can act as a 'stealth' virus to counteract the innate immune responses mediated by the pathogen recognition receptors of hepatocytes and escape the first line of surveillance of the host immune system. We give an overview of the cellular components of innate immunity that present in these in vitro models and discuss how activating these innate immunity components may contribute to the eradication of HBV infection.

Is cell culture a suitable tool for the evaluation of micro- and nanoplastics ecotoxicity?

Plastic particles have been described in aquatic ecosystems worldwide. An increasing number of studies have tried to evaluate the toxic impacts of microplastics (1-5000 µm) but also nanoplastics (<1 µm) in marine and freshwater organisms. However, the wide variety of plastic particles characteristics such as various sizes, shapes, functionalization or types of polymer, makes it difficult to evaluate their impact with regular ecotoxicity testing. In this context, cell culture, mainly used in human toxicology, could be a promising tool to evaluate micro- and nanoplastics toxicity with a wide diversity of conditions allowing to generate a large set of data. This review presents the current research on micro and nanoplastics using cell culture of marine and freshwater organisms, describes the limitations of cell culture tool and defines whether this tool can be considered as a relevant alternative strategy for ecotoxic evaluation of micro and nanoplastics especially for future regulatory needs. Articles using specifically cell culture tool from aquatic organisms such as fish or bivalves were identified. The majority evaluated the toxicity of polystyrene nanobeads on immune parameters, oxidative stress or DNA damage in fish cells. Although most of the papers characterized nanoplastic particles into the cell culture media, the relevance of testing conditions is not always clear. The development of cell culture can offer many opportunities for the evaluation of plastic particles' cellular impacts, but more research is needed to develop relevant culture models, on various aquatic organisms, and with consideration of abiotic parameters especially composition of cell culture media for nanoplastic evaluation.

Advanced 3D Cell Culture Techniques in Micro-Bioreactors, Part I: A Systematic Analysis of the Literature Published between 2000 and 2020

Bioreactors have proven useful for a vast amount of applications. Besides classical large-scale bioreactors and fermenters for prokaryotic and eukaryotic organisms, micro-bioreactors, as specialized bioreactor systems, have become an invaluable tool for mammalian 3D cell cultures. In this systematic review we analyze the literature in the field of eukaryotic 3D cell culture in micro-bioreactors within the last 20 years. For this, we define complexity levels with regard to the cellular 3D microenvironment concerning cell–matrix-contact, cell–cell-contact and the number of different cell types present at the same time. Moreover, we examine the data with regard to the micro-bioreactor design including mode of cell stimulation/nutrient supply and materials used for the micro-bioreactors, the corresponding 3D cell culture techniques and the related cellular microenvironment, the cell types and in vitro models used. As a data source we used the National Library of Medicine and analyzed the studies published from 2000 to 2020.