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.

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.

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.

Freeze-drying enables delicate, heat-sensitive biomaterials to be stored in a dry form even at room temperature. However, exposure to physicochemical stress induced by freeze-drying presents challenges for maintaining material characteristics and functionality upon reconstitution, for which reason excipients are required. Although wide variety of different excipients are available for pharmaceutical applications, their protective role in the freeze-drying is not yet fully understood. In this study our aim was to use molecular dynamics simulations to screen the properties of different sugars and amino acids, which could be combined with plant-based nanofibrillated cellulose (NFC) hydrogel to provide protective matrix system for future freeze-drying for pharmaceuticals and biologics. The changes in the NFC-based formulations before and after freeze-drying and reconstitution were evaluated using non-invasive Timegate PicoRaman spectroscopy and traditional characterization methods. We continued to the freeze-drying with the NFC hydrogel formulations including lactose with and without glycine, which showed the highest attraction preferences on NFC surface in silico. This formulation enabled successful freeze-drying and subsequent reconstitution with preserved physicochemical and rheological properties. Raman spectroscopy gave us insights of the molecular-level changes during freeze-drying, especially the mutarotation of lactose. This research showed the potential of integrating in silico screening and non-invasive spectroscopical method to design novel biomaterial-based formulations for freeze-drying. The research provided insights of the molecular-level interactions and orientational changes of the excipients, which might be crucial in future freeze-drying applications of pharmaceuticals and biologics.

Freeze-Drying (FD) has become an ever more significant part of the pharmaceutical industry as the number of biopharmaceuticals has increased in recent decades. The growing number of advanced therapy medicinal products (ATMPs) creates new challenges for the FD industry as cells, virus vectors, and other complex biologicals need to be preserved in dry conditions at room temperature. Nanofibrillated cellulose hydrogel (NFCh) is a wood-derived sugar polymer-based product that is used in various pharmaceutical applications, such as wound dressings, as a three-dimensional (3D) cell culturing platform, and in tissue engineering. NFCh can also be utilized as an excipient in FD. However, NFCh requires the use of other excipients to provide a desirable reconstitution ability and sufficient residual moisture contents after the FD process. In this work, the effect of formulation on the FD-reconstitution process of NFCh with saccharose, lactose, trehalose, and glycine was studied. Raman- and near-infrared (NIR) spectroscopy were utilized as non-invasive technologies to determine molecular changes in the formulations during the FD-reconstitution process. Wide-Angle X-ray Scattering (WAXS) analyses were performed to reveal changes in crystallinity of the FD formulations. Disaccharides likely formed a two-phase system with NFCh and were found to have a clear decreasing effect on the crystallinity of the freeze-dried NFCh. Glycine as an amino acid was found to stabilize the formulation, possibly due to preferential exclusion, which was observed in NIR-spectra. In the future these NFCh formulations can be utilized in FD of complex biologicals to overcome issues regarding their storage and transportation.

Chronic wounds, particularly diabetic foot ulcers, heal unpredictably and impose substantial healthcare costs. Deep learning (DL) systems that integrate complementary data streams could support preclinical studies by assessing wound status and therapy efficacy reliably in small-n studies. We developed a three-branch neural network combining convolutional feature extraction for red-green-blue (RGB) images, infrared thermography, and wound-area measurements to classify healing stage and infer treatment response in diabetic mouse wound model. In descriptive ablation analysis, the model integrating RGB and wound-size inputs achieved higher accuracy than the RGB-only configuration (0.75 vs. 0.70), suggesting that explicit size context provided useful complementary information. Thermal images appeared qualitatively informative during early healing but contributed limited additional signal later, likely reflecting subtle inflammatory gradients or angiogenesis in healthy murine skin; this observation motivated a constrained weighted-fusion strategy. In the development-stage comparison, static fusion with tunable fixed weights yielded the highest observed performance (∼0.85 accuracy on the development test subject). Grad-CAM visualizations showed that predictions arose from biologically relevant regions rather than image artifacts. Experiments without subject-level partitioning produced inflated metrics, underscoring the need for grouping by subject to ensure generalizable evaluation. Nested Leave-One-Group-Out cross-validation with KerasTuner-based hyperparameter tuning yielded a mean accuracy of 0.71, estimating performance on unseen subjects. This resulting proof-of-concept model predicts treatment responses for wounds treated with nanofibrillated-cellulose hydrogel. Multimodal DL combining standard and thermal photography with size metrics can stage wound healing and detect divergent treatment trajectories and thus be used in preclinical efficacy evaluation of novel wound treatments. With larger, clinically annotated datasets, this approach could be applied in a clinical setting as well.