Assessing cell migration in hydrogels: An overview of relevant materials and methods

Cell migration is essential in numerous living processes, including embryonic development, wound healing, immune responses, and cancer metastasis. From individual cells to collectively migrating epithelial sheets, the locomotion of cells is tightly regulated by multiple structural, chemical, and biological factors. However, the high complexity of this process limits the understanding of the influence of each factor. Recent advances in materials science, tissue engineering, and microtechnology have expanded the toolbox and allowed the development of biomimetic in vitro assays to investigate the mechanisms of cell migration. Particularly, three-dimensional (3D) hydrogels have demonstrated a superior ability to mimic the extracellular environment. They are therefore well suited to studying cell migration in a physiologically relevant and more straightforward manner than in vivo approaches. A myriad of synthetic and naturally derived hydrogels with heterogeneous characteristics and functional properties have been reported. The extensive portfolio of available hydrogels with different mechanical and biological properties can trigger distinct biological responses in cells affecting their locomotion dynamics in 3D. Herein, we describe the most relevant hydrogels and their associated physico-chemical characteristics typically employed to study cell migration, including established cell migration assays and tracking methods. We aim to give the reader insight into existing literature and practical details necessary for performing cell migration studies in 3D environments.publishedVersio

Migration of T-cells in a multi-compartment hydrogel model of inflammation

In the present work multi-compartmental hydrogels were established in order to study T-cell migration in 3D. The main approach that was developed for the fabrication of the hydrogel systems, was based on the incorporation of an alginate compartment with an encapsulated bioactive factor, in a surrounding 3D matrix composed of collagen type-I, Matrigel or chemically modified alginate. The resulted hydrogels were used as 3D in vitro platforms, in order to evaluate the migration of T-cells under the influence of pro-inflammatory mediators secreted by stimulated dendritic cells (DCs). DCs where encapsulated into the alginate together with lipopolysaccharide LPS that resulted to the activation of the TLR4 pathway, followed by the secretion of pro-inflammatory mediators. The stimulation of the DCs regulated the migration of the T-cell hybridomas embedded in the surrounding scaffolds. The multi-compartmental hydrogel system was successfully used to demonstrate migration in collagen type-I and Matrigel. The activity of T-cell hybridomas in response to pro-inflammatory mediators was monitored in fibrillar collagen type-I matrix for the timepoints of 0h, 4h, 6h, 8h, 24h and 48h after the system was assembled, with live cell imaging. The results suggested an elevated accumulation around the alginate compartment throughout time in respect to the inflamed microenvironment that was exponentially increased at 48h. The migration in collagen-type I was in addition quantitated by automated cell tracking at the aforementioned timepoints. No significant differences were observed in terms of migrated distances and cell speed during the first 24h while at 48h the values were significantly increased. In general, the results indicated an increased migration of T-cell hybridomas under the influence of pro-inflammatory mediators with most critical timepoint that of 48h. The motility behavior of T-cell hybridomas was analyzed by mean squared displacement analysis and further discussed. For multi-compartmental hydrogels where Matrigel was employed as a surrounding scaffold imaging took place at 0h and 96h. The T-cell hybridomas were shown to conglomerate forming a distinguishable line at the edge of the alginate structure at 96h under the presence of activated DCs. The results have indicated a directional migration to the endotoxin stimulated source. The same system was assessed in chemically modified alginates as a surrounding matrix. No activity was demonstrated in sulfated and partially oxidized-reduced (POAred) alginate hydrogels. Pilot experiments were conducted in RGD-alginate hydrogels of different weight average molecular weight (Mw) compositions. The migration of T-cell hybridomas was recorded in both and Mw compositions. After one day of incubation the migration was increased within the hydrogels of both Mw compositions. The increased motility in the scaffolds after one day could be relevant to transitions that took place in the alginate network and altered the hydrogel architecture. Lastly, a comparison in T-cell hybridomas morphology during migration in the different biomaterial scaffolds. Cell morphology was found to meet different attributes between the different biomaterials and could be linked to the surrounding matrix architecture. In conclusion, multi-compartmental hydrogels based on fibrillar collagen and Matrigel with encapsulated alginate microstructures were successfully produced as an in vitro platform in order to study T-cell migration in 3D under the influence of an innate immune response. Alginate can be a good surrounding scaffold candidate in future setups since migration of T-cell hybridomas was determined within the hydrogels

Enhancing pre-clinical research with simplified intestinal cell line models

Two-dimensional culture remains widely employed to determine the bioavailability of orally delivered drugs. To gain more knowledge about drug uptake mechanisms and risk assessment for the patient after oral drug admission, intestinal in vitro models demonstrating a closer similarity to the in vivo situation are needed. In particular, Caco-2 cell-based Transwell® models show advantages as they are reproducible, cost-efficient, and standardized. However, cellular complexity is impaired and cell function is strongly modified as important transporters in the apical membrane are missing. To overcome these limitations, primary organoid-based human small intestinal tissue models were developed recently but the application of these cultures in pre-clinical research still represents an enormous challenge, as culture setup is complex as well as time- and cost-intensive. To overcome these hurdles, we demonstrate the establishment of primary organoid-derived intestinal cell lines by immortalization. Besides exhibiting cellular diversity of the organoid, these immortalized cell lines enable a standardized and more cost-efficient culture. Further, our cell line-based Transwell®-like models display an organ-specific epithelial barrier integrity, ultrastructural features and representative transport functions. Altogether, our novel model systems are cost-efficient with close similarity to the in vivo situation, therefore favoring their use in bioavailability studies in the context of pre-clinical screenings

sj-docx-1-tej-10.1177_20417314241228949 – Supplemental material for Enhancing pre-clinical research with simplified intestinal cell line models

Supplemental material, sj-docx-1-tej-10.1177_20417314241228949 for Enhancing pre-clinical research with simplified intestinal cell line models by Christina Fey, Theresa Truschel, Kristina Nehlsen, Spyridon Damigos, Julia Horstmann, Theresia Stradal, Tobias May, Marco Metzger and Daniela Zdzieblo in Journal of Tissue Engineering</p