Marker-controlled watershed with deep edge emphasis and optimized H-minima transform for automatic segmentation of densely cultivated 3D cell nuclei.

BACKGROUND: The segmentation of 3D cell nuclei is essential in many tasks, such as targeted molecular radiotherapies (MRT) for metastatic tumours, toxicity screening, and the observation of proliferating cells. In recent years, one popular method for automatic segmentation of nuclei has been deep learning enhanced marker-controlled watershed transform. In this method, convolutional neural networks (CNNs) have been used to create nuclei masks and markers, and the watershed algorithm for the instance segmentation. We studied whether this method could be improved for the segmentation of densely cultivated 3D nuclei via developing multiple system configurations in which we studied the effect of edge emphasizing CNNs, and optimized H-minima transform for mask and marker generation, respectively. RESULTS: The dataset used for training and evaluation consisted of twelve in vitro cultivated densely packed 3D human carcinoma cell spheroids imaged using a confocal microscope. With this dataset, the evaluation was performed using a cross-validation scheme. In addition, four independent datasets were used for evaluation. The datasets were resampled near isotropic for our experiments. The baseline deep learning enhanced marker-controlled watershed obtained an average of 0.69 Panoptic Quality (PQ) and 0.66 Aggregated Jaccard Index (AJI) over the twelve spheroids. Using a system configuration, which was otherwise the same but used 3D-based edge emphasizing CNNs and optimized H-minima transform, the scores increased to 0.76 and 0.77, respectively. When using the independent datasets for evaluation, the best performing system configuration was shown to outperform or equal the baseline and a set of well-known cell segmentation approaches. CONCLUSIONS: The use of edge emphasizing U-Nets and optimized H-minima transform can improve the marker-controlled watershed transform for segmentation of densely cultivated 3D cell nuclei. A novel dataset of twelve spheroids was introduced to the public.

Label-Free Analysis with Multiple Parameters Separates G Protein-Coupled Receptor Signaling Pathways.

Real-time label-free techniques are used to profile G protein-coupled receptor (GPCR) signaling pathways in living cells. However, interpreting the label-free signal responses is challenging, and previously reported methods do not reliably separate pathways from each other. In this study, a continuous angular-scanning surface plasmon resonance (SPR) technique is utilized for measuring label-free GPCR signal profiles. We show how the continuous angular-scanning ability, measuring up to nine real-time label-free parameters simultaneously, results in more information-rich label-free signal profiles for different GPCR pathways, providing a more accurate pathway separation. For this, we measured real-time full-angular SPR response curves for Gs, Gq, and Gi signaling pathways in living cells. By selecting two of the most prominent label-free parameters: the full SPR curve angular and intensity shifts, we present how this analysis approach can separate each of the three signaling pathways in a straightforward single-step analysis setup, without concurrent use of signal inhibitors or other response modulating compounds.

Effects of nanofibrillated cellulose hydrogels on adipose tissue extract and hepatocellular carcinoma cell spheroids in freeze-drying.

The aim of this study was to evaluate the effects of two nanofibrillated cellulose (NFC) hydrogels on two human derivatives during freeze-drying. Native NFC hydrogel is a suitable platform to culture 3D cell spheroids and a hydrogel processed further, called anionic NFC (ANFC) hydrogel, is an excellent platform for controlled release of proteins. Moreover, it has been shown to be compatible with freeze-drying when correct lyoprotectants are implemented. Freeze-drying is a method, where substance is first frozen, and then vacuum dried trough sublimation of water in order to achieve dry matter without the loss of the original three-dimensional structures. The first chosen human derivative was adipose tissue extract (ATE) which is a cell-free growth factor-rich preparation capable of promoting growth of regenerative cells. The release of growth factors from the freeze-dried mixture of ATE and ANFC was compared to that of non-freeze-dried control mixtures. The release profiles remained at the same level after freeze-drying. The second derivative was hepatocellular carcinoma (HepG2) cell spheroids which were evaluated before and after freeze-drying. The 3D structure of the HepG2 cell spheroids was preserved and the spheroids retained 18% of their metabolic activity after rehydration. However, the freeze-dried and rehydrated HepG2 cell spheroids did not proliferate and the cell membrane was damaged by fusion and formation of crystals.

Barcode lipids for absolute quantitation of liposomes in ocular tissues.

Lipid-based drug formulations are promising systems for improving delivery of drugs to ocular tissues, such as retina. To develop lipid-based systems further, an improved understanding of their pharmacokinetics is required, but high-quality in vivo experiments require a large number of animals, raising ethical and economic questions. In order to expedite in vivo kinetic testing of lipid-based systems, we propose a barcode approach that is based on barcoding liposomes with non-endogenous lipids. We developed and evaluated a liquid-chromatography-mass spectrometry method to quantify many liposomes simultaneously in aqueous humor, vitreous, and neural retina at higher than ±20% precision and accuracy. Furthermore, we showed in vivo suitability of the method in pharmacokinetic evaluation of six different liposomes after their simultaneous injection into the rat vitreal cavity. We calculated pharmacokinetic parameters in vitreous and aqueous humor, quantified liposome concentrations in the retina, and quantitated retinal distribution of the liposomes in the rats. Compared to individual injections of the liposome formulations, the barcode-based study design enabled reduction of animal numbers from 72 to 12. We believe that the proposed approach is reliable and will reduce and refine ocular pharmacokinetic experiments with liposomes and other lipid-based systems.

Stiffness-Controlled Hydrogels for 3D Cell Culture Models.

Nanofibrillated cellulose (NFC) hydrogel is a versatile biomaterial suitable, for example, for three-dimensional (3D) cell spheroid culturing, drug delivery, and wound treatment. By freeze-drying NFC hydrogel, highly porous NFC structures can be manufactured. We freeze-dried NFC hydrogel and subsequently reconstituted the samples into a variety of concentrations of NFC fibers, which resulted in different stiffness of the material, i.e., different mechanical cues. After the successful freeze-drying and reconstitution, we showed that freeze-dried NFC hydrogel can be used for one-step 3D cell spheroid culturing of primary mesenchymal stem/stromal cells, prostate cancer cells (PC3), and hepatocellular carcinoma cells (HepG2). No difference was observed in the viability or morphology between the 3D cell spheroids cultured in the freeze-dried and reconstituted NFC hydrogel and fresh NFC hydrogel. Furthermore, the 3D cultured spheroids showed stable metabolic activity and nearly 100% viability. Finally, we applied a convolutional neural network (CNN)-based automatic nuclei segmentation approach to automatically segment individual cells of 3D cultured PC3 and HepG2 spheroids. These results provide an application to culture 3D cell spheroids more readily with the NFC hydrogel and a step towards automatization of 3D cell culturing and analysis.

Angiogenic Potential of Human Adipose-Derived Mesenchymal Stromal Cells in Nanofibrillated Cellulose Hydrogel.

Adipose-derived mesenchymal stromal cells (ASCs) hold great potential for cellular therapies by having immunomodulatory behavior and tissue regenerative properties. Due to the capability of ASCs to differentiate into endothelial cells (ECs) and other angiogenic cell types, such as pericytes, ASCs are a highly valuable source for stimulating angiogenesis. However, cellular therapies in tissue engineering have faced challenges in poor survival of the cells after transplantation, which is why a protective biomaterial scaffold is required. In this work, we studied the potential of nanofibrillated cellulose (NFC) hydrogel to be utilized as a suitable matrix for three-dimensional (3D) cell culturing of human-derived ASCs (hASCs) and studied their angiogenic properties and differentiation potential in ECs and pericytes. In addition, we tested the effect of hASC-conditioned medium and stimulation with angiopoietin-1 (Ang-1) on human umbilical vein endothelial cells (HUVECs) to induce blood vessel-type tube formation in NFC hydrogel. The hASCs were successfully 3D cell cultured in NFC hydrogel as they formed spheroids and had high cell viability with angiogenic features. Most importantly, they showed angiogenic potential by having pericyte-like characteristics when differentiated in EC medium, and their conditioned medium improved HUVEC viability and tube formation, which recalls the active paracrine properties. This study recommends NFC hydrogel for future use as an animal-free biomaterial scaffold for hASCs in therapeutic angiogenesis and other cell therapy purposes.

Near-infrared analysis of nanofibrillated cellulose aerogel manufacturing.

Biomaterial aerogel fabrication by freeze-drying must be further improved to reduce the costs of lengthy freeze-drying cycles and to avoid the formation of spongy cryogels and collapse of the aerogel structures. Residual water content is a critical quality attribute of the freeze-dried product, which can be monitored in-line with near-infrared (NIR) spectroscopy. Predictive models of NIR have not been previously applied for biomaterials and the models were mostly focused on the prediction of only one formulation at a time. We recorded NIR spectra of different nanofibrillated cellulose (NFC) hydrogel formulations during the secondary drying and set up a partial least square regression model to predict their residual water contents. The model can be generalized to measure residual water of formulations with different NFC concentrations and the excipients, and the NFC fiber concentrations and excipients can be separated with the principal component analysis. Our results provide valuable information about the freeze-drying of biomaterials and aerogel fabrication, and how NIR spectroscopy can be utilized in the optimization of residual water content.

Preservation of biomaterials and cells by freeze-drying: Change of paradigm.

Freeze-drying is the most widespread method to preserve protein drugs and vaccines in a dry form facilitating their storage and transportation without the laborious and expensive cold chain. Extending this method for the preservation of natural biomaterials and cells in a dry form would provide similar benefits, but most results in the domain are still below expectations. In this review, rather than consider freeze-drying as a traditional black box we "break it" through a detailed process thinking approach. We discuss freeze-drying from process thinking aspects, introduce the chemical, physical, and mechanical environments important in this process, and present advanced biophotonic process analytical technology. In the end, we review the state of the art in the freeze-drying of the biomaterials, extracellular vesicles, and cells. We suggest that the rational design of the experiment and implementation of advanced biophotonic tools are required to successfully preserve the natural biomaterials and cells by freeze-drying. We discuss this change of paradigm with existing literature and elaborate on our perspective based on our new unpublished results.

Molecular Insights on Successful Reconstitution of Freeze-Dried Nanofibrillated Cellulose Hydrogel.

The diversity and safety of nanofibrillated cellulose (NFC) hydrogels have gained a vast amount of interest at the pharmaceutical site in recent years. Moreover, this biomaterial has a high potential to be utilized as a protective matrix during the freeze-drying of heat-sensitive pharmaceuticals and biologics to increase their properties for long-term storing at room temperature and transportation. Since freeze-drying and subsequent reconstitution have not been optimized for this biomaterial, we must find a wider understanding of the process itself as well as the molecular level interactions between the NFC hydrogel and the most suitable lyoprotectants. Herein we optimized the reconstitution of the freeze-dried NFC hydrogel by considering critical quality attributes required to ensure the success of the process and gained insights of the obtained experimental data by simulating the effects of the used lyoprotectants on water and NFC. We discovered the correlation between the measured characteristics and molecular dynamics simulations and obtained successful freeze-drying and subsequent reconstitution of NFC hydrogel with the presence of 300 mM of sucrose. These findings demonstrated the possibility of using the simulations together with the experimental measurements to obtain a more comprehensive way to design a successful freeze-drying process, which could be utilized in future pharmaceutical applications.

Nanofibrillar cellulose wound dressing supports the growth and characteristics of human mesenchymal stem/stromal cells without cell adhesion coatings.

BACKGROUND: In the field of regenerative medicine, delivery of human adipose-derived mesenchymal stem/stromal cells (hASCs) has shown great promise to promote wound healing. However, a hostile environment of the injured tissue has shown considerably to limit the survival rate of the transplanted cells, and thus, to improve the cell survival and retention towards successful cell transplantation, an optimal cell scaffold is required. The objective of this study was to evaluate the potential use of wood-derived nanofibrillar cellulose (NFC) wound dressing as a cell scaffold material for hASCs in order to develop a cell transplantation method free from animal-derived components for wound treatment. METHODS: Patient-derived hASCs were cultured on NFC wound dressing without cell adhesion coatings. Cell characteristics, including cell viability, morphology, cytoskeletal structure, proliferation potency, and mesenchymal cell and differentiation marker expression, were analyzed using cell viability assays, electron microscopy, immunocytochemistry, and quantitative or reverse transcriptase PCR. Student's t test and one-way ANOVA followed by a Tukey honestly significant difference post hoc test were used to determine statistical significance. RESULTS: hASCs were able to adhere to NFC dressing and maintained high cell survival without cell adhesion coatings with a cell density-dependent manner for the studied period of 2 weeks. In addition, NFC dressing did not induce any remarkable cytotoxicity towards hASCs or alter the morphology, proliferation potency, filamentous actin structure, the expression of mesenchymal vimentin and extracellular matrix (ECM) proteins collagen I and fibronectin, or the undifferentiated state of hASCs. CONCLUSIONS: As a result, NFC wound dressing offers a functional cell culture platform for hASCs to be used further for in vivo wound healing studies in the future.