Microarray Platforms Based on 3D Printing.

This paper introduces an innovative method for the fabrication and infusion of microwell arrays based on digital light processing (DLP) 3D printing. A low-cost DLP 3D printer is employed to fabricate microstructures rapidly with a broad dynamic range while maintaining high precision and fidelity. We constructed microwell arrays with varying diameters, from 200 to 2000 µm and multiple aspect ratios, in addition to microchannels with widths ranging from 45 to 1000 µm, proving the potential and flexibility of this fabrication method. The superimposition of parallel microchannels onto the microwell array, facilitated by positive or negative pressure, enabled the transfer of liquid to the microwells. Upon removal of the microchannel chip, a dispensed microdroplet array was obtained. This array can be modulated by adjusting the volume of the microwells and the inflow fluid. The filled microwell array allows chip-to-chip dispensing to the microreactor array through binding and centrifugation, facilitating multistep and multireagent assays. The 3D printing approach also enables the fabrication of intricate cavity designs, such as micropyramid arrays, which can be integrated with parallel microchannels to generate spheroid flowcells. This device demonstrated the ability to generate spheroids and manipulate their environment. We have successfully utilized precise modulation of spheroids size and performed parallel drug dose-response assays to evaluate its effectiveness. Furthermore, we managed to execute dynamic drug combinations based on a compact spheroids array, utilizing two orthogonal parallel microchannels. Our findings suggest that both the combination and temporal sequence of drug administration have a significant impact on therapeutic outcomes.

Fluid-Dynamic Culture of Tumour and Immune Cells for More Predictive Infiltration Studies and Immunotherapy Drug Screening.

Immunotherapies represent one of the current most promising challenges in cancer treatment. They are based on the boost of natural immune responses, aimed at cancer eradication. However, the success of immunotherapeutic approaches strictly depends on the interaction between immune cells and cancer cells. Preclinical drug tests currently available are poor in fully predicting the actual safety and efficacy of immunotherapeutic treatments under development. Indeed, conventional 2D cell culture underrepresents the complexity of the tumour microenvironment, while in vivo animal models lack in mimicking the human immune cell responses. In this context, predictability, reliability, and complete immune compatibility still represent challenges to overcome. For this aim, novel 3D, fully humanized in vitro cancer tissue models have been recently optimized by adopting emerging technologies, such as organ-on-chips (OOC) and 3D cancer cell-laden hydrogels. In particular, a novel multi-in vitro organ (MIVO) OOC platform has been recently adopted to culture 3D clinically relevant size cancer tissues under proper physiological culture conditions to investigate anti-cancer treatments and immune-tumour cell crosstalk.The proposed immune-tumour OOC-based model offers a potential tool for accurately modelling human immune-related diseases and effectively assessing immunotherapy efficacy, finally offering promising experimental approaches for personalized medicine.

Biocompatibility of Synechococcus sp. PCC 7002 with Human Dermal Cells In Vitro.

Being the green gold of the future, cyanobacteria have recently attracted considerable interest worldwide. This study investigates the adaptability and biocompatibility of the cyanobacterial strain Synechococcus sp. PCC 7002 with human dermal cells, focusing on its potential application in biomedical contexts. First, we investigated the adaptability of Synechococcus PCC 7002 bacteria to human cell culture conditions. Next, we evaluated the biocompatibility of cyanobacteria with common dermal cells, like 3T3 fibroblasts and HaCaT keratinocytes. Therefore, cells were directly and indirectly cocultured with the corresponding cells, and we measured metabolic activity (AlamarBlue assay) and proliferation (cell count and PicoGreen assay). The lactate dehydrogenase (LDH) assay was performed to determine the cytotoxic effect of cyanobacteria and their nutrition medium on human dermal cells. The cyanobacteria exhibited exponential growth under conventional human cell culture conditions, with the temperature and medium composition not affecting their viability. In addition, the effect of illumination on the proliferation capacity was investigated, showing a significant impact of light exposure on bacterial growth. The measured oxygen production under hypoxic conditions demonstrated a sufficient oxygen supply for further tissue engineering approaches depending on the number of bacteria. There were no significant adverse effects on human cell viability and growth under coculture conditions, whereas the LDH assay assessed signs of cytotoxicity regarding 3T3 fibroblasts after 2 days of coculturing. These negative effects were dismissed after 4 days. The findings highlight the potential of Synechococcus sp. PCC 7002 for integration into biomedical approaches. We found no cytotoxicity of cyanobacteria on 3T3 fibroblasts and HaCaT keratinocytes, thus paving the way for further in vivo studies to assess long-term effects and systemic reactions.

Understanding PFAS toxicity through cell culture metabolomics: Current applications and future perspectives.

Per- and polyfluoroalkyl substances (PFAS), ubiquitous environmental contaminants, pose significant challenges to ecosystems and human health. While cell cultures have emerged as new approach methodologies (NAMs) in ecotoxicity research, metabolomics is an emerging technique used to characterize the small-molecule metabolites present in cells and to understand their role in various biological processes. Integration of metabolomics with cell cultures, known as cell culture metabolomics, provides a novel and robust tool to unravel the complex molecular responses induced by PFAS exposure. In vitro testing also reduces reliance on animal testing, aligning with ethical and regulatory imperatives. The current review summarizes key findings from recent studies utilizing cell culture metabolomics to investigate PFAS toxicity, highlighting alterations in metabolic pathways, biomarker identification, and the potential linkages between metabolic perturbations. Additionally, the paper discusses different types of cell cultures and metabolomics methods used for studies of environmental contaminants and particularly PFAS. Future perspectives on the combination of metabolomics with other advanced technologies, such as single-cell metabolomics (SCM), imaging mass spectrometry (IMS), extracellular flux analysis (EFA), and multi-omics are also explored, which offers a holistic understanding of environmental contaminants. The synthesis of current knowledge and identification of research gaps provide a foundation for future investigations that aim to elucidate the complexities of PFAS-induced cellular responses and contribute to the development of effective strategies for mitigating their adverse effects on human health.

Proceedings of the 2023 Viral Clearance Symposium, Session 7: Up- and Downstream Virus Retentive Filtration.

Session 7 of the 2023 Viral Clearance Symposium reviewed progresses in virus retentive filtrations applied to both upstream and downstream processing. Upstream topics included investigations and applications of media viral filtration for upstream cell culture viral risk mitigation. Downstream topics included evaluation of viral breakthrough in continuous processing using surrogate particles and demonstration of extensive viral filtration cycling with flow interruptions and long duration in connected process. Reuse of viral filters with proposed procedures was successfully demonstrated amid the supply chain challenge encountered during the pandemic. Discussions and additional considerations for the topics were also provided.

A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactors.

Ex vivo genetically-modified cellular immunotherapies, such as chimeric antigen receptor T cell (CAR-T) therapies, have generated significant clinical and commercial outcomes due to their unparalleled response rates against relapsed and refractory blood cancers. However, the development and scalable manufacture of these novel therapies remains challenging and further process understanding and optimisation is required to improve product quality and yield. In this study, we employ a quality-by-design (QbD) approach to systematically investigate the impact of critical process parameters (CPPs) during the expansion step on the critical quality attributes (CQAs) of CAR-T cells. Utilising the design of experiments (DOE) methodology, we investigated the impact of multiple CPPs, such as number of activations, culture seeding density, seed train time, and IL-2 concentration, on CAR-T CQAs including, cell yield, viability, metabolism, immunophenotype, T cell differentiation, exhaustion and CAR expression. Initial studies undertaken in G-Rex® 24 multi-well plates demonstrated that the combination of a single activation step and a shorter, 3-day, seed train resulted in significant CAR-T yield and quality improvements, specifically a 3-fold increase in cell yield, a 30% reduction in exhaustion marker expression and more efficient metabolism when compared to a process involving 2 activation steps and a 7-day seed train. Similar findings were observed when the CPPs identified in the G-Rex® multi-well plates studies were translated to a larger-scale automated, controlled stirred-tank bioreactor (Ambr® 250 High Throughput) process. The single activation step and reduced seed train time resulted in a similar, significant improvement in CAR-T CQAs including cell yield, quality and metabolism in the Ambr® 250 High Throughput bioreactor, thereby validating the findings of the small-scale studies and resulting in significant process understanding and improvements. This study provides a methodology for the systematic investigation of CAR-T CPPs and the findings demonstrate the scope and impact of enhanced process understanding for improved CAR-T production.

Comparison of techniques for corneal epithelium cell culture for the collection of conditioned medium.

PURPOSES: To determine the best protocol in obtaining the higher yield of conditioned culture medium to be used for the bone marrow mesenchymal stem cell differentiation into corneal epithelial cells, five techniques for the primary culture of human corneal epithelial cells were evaluated. METHODS: The studied culture techniques of corneal epithelial cells were: explants in culture flasks with and without hydrophilic surface treatment, on amniotic membrane, with enzymatic digestion, and by corneal scraping. The conditioned culture medium collected from these cultures was used to differentiate human bone marrow mesenchymal stem cells into corneal epithelial cells, which were characterized using flow cytometry with pan-cytokeratin and the corneal-specific markers, cytokeratin 3 and cytokeratin 12. RESULTS: The culture technique using flasks with hydrophilic surface treatment resulted in the highest yield of conditioned culture medium. Flasks without surface treatment resulted to a very low success rate. Enzymatic digestion and corneal scraping showed contamination with corneal fibroblasts. The culture on amniotic membranes only allowed the collection of culture medium during the 1st cell confluence. The effectiveness of cell differentiation was confirmed by cytometry analysis using the collected conditioned culture medium, as demonstrated by the expressions of cytokeratin 3 (95.3%), cytokeratin 12 (93.4%), and pan-cytokeratin (95.3%). CONCLUSION: The culture of corneal epithelial cell explants in flasks with hydrophilic surface treatment is the best technique for collecting a higher yield of conditioned culture medium to be used to differentiate mesenchymal stem cells.

Explainable AI for CHO cell culture media optimization and prediction of critical quality attribute.

Cell culture media play a critical role in cell growth and propagation by providing a substrate; media components can also modulate the critical quality attributes (CQAs). However, the inherent complexity of the cell culture media makes unraveling the impact of the various media components on cell growth and CQAs non-trivial. In this study, we demonstrate an end-to-end machine learning framework for media component selection and prediction of CQAs. The preliminary dataset for feature selection was generated by performing CHO-GS (-/-) cell culture in media formulations with varying metal ion concentrations. Acidic and basic charge variant composition of the innovator product (24.97 ± 0.54% acidic and 11.41 ± 1.44% basic) was chosen as the target variable to evaluate the media formulations. Pearson's correlation coefficient and random forest-based techniques were used for feature ranking and feature selection for the prediction of acidic and basic charge variants. Furthermore, a global interpretation analysis using SHapley Additive exPlanations was utilized to select optimal features by evaluating the contributions of each feature in the extracted vectors. Finally, the medium combinations were predicted by employing fifteen different regression models and utilizing a grid search and random search cross-validation for hyperparameter optimization. Experimental results demonstrate that Fe and Zn significantly impact the charge variant profile. This study aims to offer insights that are pertinent to both innovators seeking to establish a complete pipeline for media development and optimization and biosimilar-based manufacturers who strive to demonstrate the analytical and functional biosimilarity of their products to the innovator. KEY POINTS: • Developed a framework for optimizing media components and prediction of CQA. • SHAP enhances global interpretability, aiding informed decision-making. • Fifteen regression models were employed to predict medium combinations.

Deciphering the importance of culture pH on CD22 CAR T-cells characteristics.

BACKGROUND: Chimeric antigen receptor (CAR) T-cells have demonstrated significant efficacy in targeting hematological malignancies, and their use continues to expand. Despite substantial efforts spent on the optimization of protocols for CAR T-cell manufacturing, critical parameters of cell culture such as pH or oxygenation are rarely actively monitored during cGMP CAR T-cell generation. A comprehensive understanding of the role that these factors play in manufacturing may help in optimizing patient-specific CAR T-cell therapy with maximum benefits and minimal toxicity. METHODS: This retrospective study examined cell culture supernatants from the manufacture of CAR T-cells for 20 patients with B-cell malignancies enrolled in a phase 1/2 clinical trial of anti-CD22 CAR T-cells. MetaFLEX was used to measure supernatant pH, oxygenation, and metabolites, and a Bio-Plex assay was used to assess protein levels. Correlations were assessed between the pH of cell culture media throughout manufacturing and cell proliferation as well as clinical outcomes. Next-generation sequencing was conducted to examine gene expression profiles of the final CAR T-cell products. RESULTS: A pH level at the lower range of normal at the beginning of the manufacturing process significantly correlated with measures of T-cell expansion and metabolism. Stable or rising pH during the manufacturing process was associated with clinical response, whereas a drop in pH was associated with non-response. CONCLUSIONS: pH has potential to serve as an informative factor in predicting CAR T-cell quality and clinical outcomes. Thus, its active monitoring during manufacturing may ensure a more effective CAR T-cell product.

Derivation and long-term maintenance of porcine skeletal muscle progenitor cells.

Culture of muscle cells from livestock species has typically involved laborious enzyme-based approaches that yield heterogeneous populations with limited proliferative and myogenic differentiation capacity, thus limiting their use in physiologically-meaningful studies. This study reports the use of a simple explant culture technique to derive progenitor cell populations from porcine muscle that could be maintained and differentiated long-term in culture. Fragments of semitendinosus muscle from 4 to 8 week-old piglets (n = 4) were seeded on matrigel coated culture dishes to stimulate migration of muscle-derived progenitor cells (MDPCs). Cell outgrowths appeared within a few days and were serially passaged and characterised using RT-qPCR, immunostaining and flow cytometry. MDPCs had an initial mean doubling time of 1.4 days which increased to 2.5 days by passage 14. MDPC populations displayed steady levels of the lineage-specific markers, PAX7 and MYOD, up until at least passage 2 (positive immunostaining in about 40% cells for each gene), after which the expression of myogenic markers decreased gradually. Remarkably, MDPCs were able to readily generate myotubes in culture up until passage 8. Moreover, a decrease in myogenic capacity during serial passaging was concomitant with a gradual increase in the expression of the pre-adipocyte markers, CD105 and PDGFRA, and an increase in the ability of MDPCs to differentiate into adipocytes. In conclusion, explant culture provided a simple and efficient method to harvest enriched myogenic progenitors from pig skeletal muscle which could be maintained long-term and differentiated in vitro, thus providing a suitable system for studies on porcine muscle biology and applications in the expanding field of cultured meat.

Systematic optimization and evaluation of culture conditions for the construction of circulating tumor cell clusters using breast cancer cell lines.

BACKGROUND: Circulating tumor cell (CTC) clusters play a critical role in carcinoma metastasis. However, the rarity of CTC clusters and the limitations of capture techniques have retarded the research progress. In vitro CTC clusters model can help to further understand the biological properties of CTC clusters and their clinical significance. Therefore, it is necessary to establish reliable in vitro methodological models to form CTC clusters whose biological characteristics are very similar to clinical CTC clusters. METHODS: The assays of immunofluorescence, transmission electron microscopy, EdU incorporation, cell adhension and microfluidic chips were used. The experimental metastasis model in mice was used. RESULTS: We systematically optimized the culture methods to form in vitro CTC clusters model, and more importantly, evaluated it with reference to the biological capabilities of reported clinical CTC clusters. In vitro CTC clusters exhibited a high degree of similarity to the reported pathological characteristics of CTC clusters isolated from patients at different stages of tumor metastasis, including the appearance morphology, size, adhesive and tight junctions-associated proteins, and other indicators of CTC clusters. Furthermore, in vivo experiments also demonstrated that the CTC clusters had an enhanced ability to grow and metastasize compared to single CTC. CONCLUSIONS: The study provides a reliable model to help to obtain comparatively stable and qualified CTC clusters in vitro, propelling the studies on tumor metastasis.

A high-precision wound healing assay based on photosensitized culture substrates.

Quantitative assessment of cell migration in vitro is often required in fundamental and applied research from different biomedical areas including wound repair, tumor metastasis or developmental biology. A collection of assays has been established throughout the years like the most widely used scratch assay or the so-called barrier assay. It is the principle of these assays to introduce a lesion into an otherwise confluent monolayer in order to study the migration of cells from the periphery into this artificial wound and determine the migration rate from the time necessary for wound closure. A novel assay makes use of photosensitizers doped into a polystyrene matrix. A thin layer of this composite material is coated on the bottom of regular cell culture ware showing perfect biocompatibility. When adherent cells are grown on this coating, resonant excitation of the photosensitizer induces a very local generation of 1O2, which kills the cells residing at the site of illumination. Cells outside the site of illumination are not harmed. When excitation of the photosensitizer is conducted by microscopic illumination, high-precision wounding in any size and geometry is available even in microfluidic channels. Besides proof-of-concept experiments, this study gives further insight into the mechanism of photosensitizer-mediated cell wounding.

Expression and purification of fluorinated proteins from mammalian suspension culture.

The site-specific encoding of noncanonical amino acids allows for the introduction of rationalized chemistry into a target protein. Of the methods that enable this technology, evolved tRNA and synthetase pairs offer the potential for expanded protein production and purification. Such an approach combines the versatility of solid-phase peptide synthesis with the scalable features of recombinant protein production. We describe the large scale production and purification of eukaryotic proteins bearing fluorinated phenylalanine in mammalian suspension cell preparations. Downstream applications of this approach include scalable recombinant protein preparation for ligand binding assays with small molecules and ligands, protein structure determination, and protein stability assays.

Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation.

BACKGROUND: Human pluripotent stem cells (hPSCs) have an enormous therapeutic potential, but large quantities of cells will need to be supplied by reliable, economically viable production processes. The suspension culture (three-dimensional; 3D) of hPSCs in stirred tank bioreactors (STBRs) has enormous potential for fuelling these cell demands. In this study, the efficient long-term matrix-free suspension culture of hPSC aggregates is shown. METHODS AND RESULTS: STBR-controlled, chemical aggregate dissociation and optimized passage duration of 3 or 4 days promotes exponential hPSC proliferation, process efficiency and upscaling by a seed train approach. Intermediate high-density cryopreservation of suspension-derived hPSCs followed by direct STBR inoculation enabled complete omission of matrix-dependent 2D (two-dimensional) culture. Optimized 3D cultivation over 8 passages (32 days) cumulatively yielded ≈4.7 × 1015 cells, while maintaining hPSCs' pluripotency, differentiation potential and karyotype stability. Gene expression profiling reveals novel insights into the adaption of hPSCs to continuous 3D culture compared to conventional 2D controls. CONCLUSIONS: Together, an entirely matrix-free, highly efficient, flexible and automation-friendly hPSC expansion strategy is demonstrated, facilitating the development of good manufacturing practice-compliant closed-system manufacturing in large scale.

MicroBundleCompute: Automated segmentation, tracking, and analysis of subdomain deformation in cardiac microbundles.

Advancing human induced pluripotent stem cell derived cardiomyocyte (hiPSC-CM) technology will lead to significant progress ranging from disease modeling, to drug discovery, to regenerative tissue engineering. Yet, alongside these potential opportunities comes a critical challenge: attaining mature hiPSC-CM tissues. At present, there are multiple techniques to promote maturity of hiPSC-CMs including physical platforms and cell culture protocols. However, when it comes to making quantitative comparisons of functional behavior, there are limited options for reliably and reproducibly computing functional metrics that are suitable for direct cross-system comparison. In addition, the current standard functional metrics obtained from time-lapse images of cardiac microbundle contraction reported in the field (i.e., post forces, average tissue stress) do not take full advantage of the available information present in these data (i.e., full-field tissue displacements and strains). Thus, we present "MicroBundleCompute," a computational framework for automatic quantification of morphology-based mechanical metrics from movies of cardiac microbundles. Briefly, this computational framework offers tools for automatic tissue segmentation, tracking, and analysis of brightfield and phase contrast movies of beating cardiac microbundles. It is straightforward to implement, runs without user intervention, requires minimal input parameter setting selection, and is computationally inexpensive. In this paper, we describe the methods underlying this computational framework, show the results of our extensive validation studies, and demonstrate the utility of exploring heterogeneous tissue deformations and strains as functional metrics. With this manuscript, we disseminate "MicroBundleCompute" as an open-source computational tool with the aim of making automated quantitative analysis of beating cardiac microbundles more accessible to the community.

Phenolic Determination in Proembryogenic Cell Complexes of Buckwheat Morphogenic Cell Culture with Osmium Tetroxide, Toluidine Blue O Dye, and Iron Chloride.

The study of the localization of secondary metabolites in both plants and the cell cultures on the intravital sections is hampered by the difficulty of obtaining thin, correctly oriented sections. Techniques for fixing tissues in resins allow these difficulties to be overcome. Properly selected tissue fixation techniques allow using different dyes to identify the compound of interest. In addition, some components of tissue fixation can act as fixatives and as a dye for identifying secondary metabolites. For example, osmium tetroxide, which fixes lipids in tissues, stains phenolic compounds black. This paper describes methods for the detection of phenolic compounds in morphogenic callus culture of buckwheat using osmium tetroxide, Toluidine Blue O dye, and ferric chloride as dyes in epoxy resin-embedded cell culture with double fixation of the material and when material fixed in Karnovsky's fixative.

Detecting abnormal cell behaviors from dry mass time series.

The prediction of pathological changes on single cell behaviour is a challenging task for deep learning models. Indeed, in self-supervised learning methods, no prior labels are used for the training and all of the information for event predictions are extracted from the data themselves. We present here a novel self-supervised learning model for the detection of anomalies in a given cell population, StArDusTS. Cells are monitored over time, and analysed to extract time-series of dry mass values. We assessed its performances on different cell lines, showing a precision of 96% in the automatic detection of anomalies. Additionally, anomaly detection was also associated with cell measurement errors inherent to the acquisition or analysis pipelines, leading to an improvement of the upstream methods for feature extraction. Our results pave the way to novel architectures for the continuous monitoring of cell cultures in applied research or bioproduction applications, and for the prediction of pathological cellular changes.

Characterization of cellular responses and cell lysis to elevated hydrodynamic stress from benchtop perfusion bioreactors.

The effective design of perfusion cell culture is currently challenging regarding balancing the operating parameters associated with the hydrodynamic conditions due to increased system complexity. To address this issue, cellular responses of an industrial CHO cell line to different types of hydrodynamic stress in benchtop perfusion bioreactors originating from agitation, sparging, and hollow fibers (HF) in the cell retention devices were systematically investigated here with the analysis of cell lysis. It was found that cell lysis was very common and most associated with the sparging stress, followed by the HF and lastly the agitation, consequently heavily impacting the estimation of process descriptors related to biomass. The results indicated that the agitation stress led to a reduced cell growth with a shift toward a more productive phenotype, suggesting an energy redirection from biomass formation to product synthesis, whereas the sparging stress had a small impact on the intracellular metabolic flux distribution but increased the cell death rate drastically. For HF stress, a similar cell maintenance profile was found as the sparging while the activity of glycolysis and the TCA cycle was significantly impeded, potentially leading to the lack of energy and thus a substantial decrease in cell-specific productivity. Moreover, a novel concept of volume average shear stress was developed to further understand the relations of different types of stress and the observed responses for an improved insight for the perfusion cell culture.

An innovative hybrid modeling approach for simultaneous prediction of cell culture process dynamics and product quality.

The use of hybrid models is extensively described in the literature to predict the process evolution in cell cultures. These models combine mechanistic and machine learning methods, allowing the prediction of complex process behavior, in the presence of many process variables, without the need to collect a large amount of data. Hybrid models cannot be directly used to predict final product critical quality attributes, or CQAs, because they are usually measured only at the end of the process, and more mechanistic knowledge is needed for many classes of CQAs. The historical models can instead predict the CQAs better; however, they cannot directly relate manipulated process parameters to final CQAs, as they require knowledge of the process evolution. In this work, we propose an innovative modeling approach based on combining a hybrid propagation model with a historical data-driven model, that is, the combined hybrid model, for simultaneous prediction of full process dynamics and CQAs. The performance of the combined hybrid model was evaluated on an industrial dataset and compared to classical black-box models, which directly relate manipulated process parameters to CQAs. The proposed combined hybrid model outperforms the black-box model by 33% on average in predicting the CQAs while requiring only around half of the data for model training to match performance. Thus, in terms of model accuracy and experimental costs, the combined hybrid model in this study provides a promising platform for process optimization applications.

Low-cost, versatile, and highly reproducible microfabrication pipeline to generate 3D-printed customised cell culture devices with complex designs.

Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices, and microfluidic systems to larger constructs such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques, equipment and tools required for their manufacture, and the expertise necessary, biological and biomedical labs tend more often to rely on already made devices. Indeed, commercially developed devices are available for a variety of applications but are often costly and, importantly, lack the potential for customisation by each individual lab. The last point is quite crucial, as often experiments in wet labs are adapted to whichever design is already available rather than designing and fabricating custom systems that perfectly fit the biological question. This combination of factors still restricts widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent advances in bioengineering and microfabrication aimed at solving these issues, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised a low-cost and highly reproducible microfabrication pipeline. This is thought specifically for biomedical and biological wet labs with not prior experience in the field, which will enable them to generate a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with limited expertise in those techniques and enables the manufacture of complex devices across the µm to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D-printed constructs for PDMS curing, using a combination of polymerisation steps, washes, and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilisation of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multipart hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.