All metabolically active tissues have a dense vascular network to supply oxygen and nutrients. Angiogenesis, the formation of new blood vessels from existing ones, can be activated by the lack of oxygen in cells, disease, trauma, or tumor growth. Due to its essential role in cell survival, angiogenesis has been extensively studied, and therapeutic control of this process is of growing interest. Simultaneously, research aims to find and standardize non-animal testing methods for future use. However, the establishment of reproducible and physiologically relevant vascular networks in vitro is challenging, particularly due to the widespread use of heterogeneous animal-derived matrices which are not in keeping with the principles of the 3Rs (Replacement, Reduction, Refinement) in science and research. To address these limitations and support the development of ethical and sustainable in vitro methods, we present a novel animal-free, three-dimensional angiogenesis model based on medical grade plant-derived nanofibrillated cellulose hydrogel (NFCh). The model uses human umbilical vein endothelial cells (HUVECs) and human adipose-derived stromal cells (hASCs) cultured in medium supplemented with human serum and NFCh matrix. First, HUVECs were stimulated with stem cell-conditioned medium and cultured in varying NFCh concentrations (0.125%-2.4%) tuned to identify the optimal matrix stiffness for capillary-like structure formation. HUVECs were then cocultured with hASCs in the best-performing hydrogel concentration, followed by imaging and proteomics analysis. We observed formation of capillary-like structures, comparable to the most commonly used animal-derived extracellular matrix (ECM) control. Interestingly cells in lower NFCh concentration (0.125%) had higher expression of wound healing and angiogenesis-related proteins while higher stiffnesses (1.5%) had higher ECM-related protein expression. Importantly, vascular network morphology and organization could be adjusted by modifying only NFCh concentration. These findings demonstrate the potential of NFCh as a reproducible, animal-free alternative to conventional angiogenesis matrices with relevance to regenerative therapies, toxicological testing, drug screening, and the development of a 3Rs-compliant in vitro system.

Platelet-rich plasma (PRP) is a source of growth factors, which are implicated in active tissue regeneration. However, after transplantation the efficacy of these bioactive compounds is often diminished due to rapid degradation and untargeted localization. For this reason, we evaluated the potential of nanofibrillated cellulose (NFC) hydrogel as a PRP carrier. NFC hydrogel is an animal-free biomaterial that, when doped with cellulase, can assist the release of PRP in a wound site. In this study, we examined the effects of 0.5% (m/v) NFC hydrogel formulations, including PRP and cellulase, on the migration and proliferation of skin cells via an in vitro scratch wound model. The suitability of the 0.8% NFC hydrogel formulations for accelerated wound healing and PRP carrying was studied in vitro in diffusion studies and in vivo in a full-thickness excisional wound model in SKH1 mice. None of the NFC hydrogel formulations with or without PRP and cellulase disturbed the normal cell behavior in vitro, and cellulase was successfully used to degrade NFC. NFC hydrogel slowed fibroblast migration rate in vitro. In vivo, NFC hydrogel treatment showed significantly enhanced re-epithelialization compared to control and supported collagen deposition. In addition, angiogenesis was significantly induced via PRP release after degrading NFC hydrogel with cellulase without abnormal host reaction. This study demonstrates the potential of NFC hydrogel with cellulase as a carrier for PRP with controlled release in future skin tissue engineering applications.

Introduction: New biologically relevant materials are needed as scaffolds for human cells in drug development, tissue models and regenerative medicine. Currently there is wide transition from animal-derived materials to synthetic and other natural ones. Wood-derived nanofibrillated cellulose (NFC) hydrogel is highly biocompatible and inert material that has been shown to provide an effective support matrix for culturing various cell types in vitro in 3D. NFC has been shown to be biocompatible also in vivo. As an animal-free matrix, NFC enables possibilities in applications aiming for clinical use, such as stem cell proliferation, organoids, and wound care. Materials and Results: (A) Pluripotent stem cells were embedded in NFC hydrogel and cultured up to 26 days. Pluripotency was analysed with OCT4 and SSEA-4 marker expression, in vitro EB-mediated differentiation, and teratoma assay. Cells proliferated in NFC without feeder cells, formed spheroids with 100-200 µm diameter, and the cells retained their pluripotency without changes in karyotypes. (B) Renal organoids can mimic the structure and function of in vivo kidneys. Organoids were cultured from primary mouse embryonic kidney metanephric mesenchymal cells followed by chemical induction to undergo nephrogenesis. Cells were embedded in NFC which reduced the distortion or stress-induced effects during the nephrogenesis process. This allowed the organoids to grow in conditions mimicking better the natural physiological environment. (C) Wound healing is a complex and continuous process which involves a variety of cells, soluble factors, and extracellular matrices. The potential of NFC for wound treatment was studied as hydrogel-based dressing and hydrogels. In addition, its potential as a cell scaffold for human adipose-derived mesenchymal stromal cells (hASCs) was studied. Clinically, NFC wound dressing has been shown to provide efficient wound healing at skin graft donor sites, and as hydrogel it did not affect wound closure rate in vivo or altered a normal healing process. Foreign-body reaction was also not observed. ASCs cultured on top of NFC dressing maintained their undifferentiated state, immunological properties and high cell viability. These findings offer a good platform to continue the development of the cell-based wound treatment. Conclusion: Nanofibrillated cellulose is a highly biocompatible material that offers well-defined matrix for various cell types in vitro and enables opportunities also in regenerative medicine applications, such as cell transplantations, due to its excellent physical properties. Authors would like to thank Yan-Ru Lou, Ulla Saarela, and Jasmi Snirvi for performing the experiments. References: 1. Lou Y. et al., 2014. The use of Nanofibrillar cellulose hydrogel as a flexible three-dimensional model to culture human pluripotent stem cells, Stem cell and development. Vol. 23:4, pp. 380–392. 2. GrowDex Application note #38 Nephrogenesis in renal organoids demonstrated in nanofibrillar cellulose hydrogel 3. Kiiskinen et al., 2019, Nanofibrillar cellulose wound dressing supports the growth and characteristics of human mesenchymal stem/stromal cells without cell adhesion coatings

Central to the success of functional precision medicine of solid tumors is to perform drug testing of patient-derived cancer cells (PDCs) in tumor-mimicking ex vivo conditions. While high throughput (HT) drug screening methods have been well-established for cells cultured in two-dimensional (2D) format, this approach may have limited value in predicting clinical responses. Here, we describe the results of the optimization of drug sensitivity and resistance testing (DSRT) in three-dimensional (3D) growth supporting matrices in a HT mode (3D-DSRT) using the hepatocyte cell line (HepG2) as an example. Supporting matrices included widely used animal-derived Matrigel and cellulose-based hydrogel, GrowDex, which has earlier been shown to support 3D growth of cell lines and stem cells. Further, the sensitivity of ovarian cancer PDCs, from two patients included in the functional precision medicine study, was tested for 52 drugs in 5 different concentrations using 3D-DSRT. Shortly, in the optimized protocol, the PDCs are embedded with matrices and seeded to 384-well plates to allow the formation of the spheroids prior to the addition of drugs in nanoliter volumes with acoustic dispenser. The sensitivity of spheroids to drug treatments is measured with cell viability readout (here, 72 h after addition of drugs). The quality control and data analysis are performed with openly available Breeze software. We show the usability of both matrices in established 3D-DSRT, and report 2D vs 3D growth condition dependent differences in sensitivities of ovarian cancer PDCs to MEK-inhibitors and cytotoxic drugs. This study provides a proof-of-concept for robust and fast screening of drug sensitivities of PDCs in 3D-DSRT, which is important not only for drug discovery but also for personalized ex vivo drug testing in functional precision medicine studies. These findings suggest that comparing results of 2D- and 3D-DSRT is essential for understanding drug mechanisms and for selecting the most effective treatment for the patient.

Phage therapy is one alternative to cure infections caused by antibiotic resistant bacteria. Due to the narrow host range of phages, hundreds to thousands of phages are required to cover the diversity of bacterial pathogens. In personalized phage therapy, fast selection of the phages for individual patients is essential for successful therapy. The aims of this study were to set up a rapid hydrogel-based liquid phage susceptibility assay (PST) for the selection of phages for therapeutic use and to establish a “ready-to-screen” plate concept, where phages are readily stored in hydrogel as small droplets in microtiter plate wells. We first tested four commercially available hydrogels (GrowDex, Askina, Purilon, and Intrasite) for their suitability as phage matrices in PSTs with four phages, two of which infecting Escherichia coli and two Staphylococcus aureus. Of these four hydrogels, GrowDex was the best matrix for PST, as it did not inhibit bacterial growth, released phages quickly when mixed with bacterial culture, and maintained phage viability well. We then optimized the assay for both optical density and microscopy readers using GrowDex as matrix with 23 bacterial strains representing 10 different species and 23 phages possessing different morphologies and genome sizes. When the bacterial growth was monitored by microscopy reader, the PST was executed in just 3 hours, and there was no need for overnight culturing bacterial cells prior to the assay, whereas using optical density reader, bacteria had to be pre-cultured overnight, and the assay time was five hours. Finally, we evaluated the effect of three different chemical stabilizers (trehalose, hyaluronic acid, and gelatin) in a six-month stability assay with six model phages. These phages assay behaved very differently in respect to the chemical stabilizers, and there was not a single stabilizer suitable for all phages. However, when gelatin (0.01%) or hyaluronic acid (0.2 mg/ml) was used as stabilizer, all tested phages were still considered as positives in PST after a six-month storage in 1 ml volume. In “ready-to-screen” plates, the differences in phage stabilities were even more profound, varying from two to six months for the most and least stable phages, respectively.

In recent times, homotypic and heterotypic 3D tumor spheroid (HTS) models have been receiving increasing attention and come to be widely employed in preclinical studies. The present study is focused on the generation of homotypic (A549 and MDA-MB-231, separately) and heterotypic (A549 + NIH/3T3; MDA-MB-231 + NIH/3T3) 3D tumor spheroids by using GrowDex® nanofibrillar cellulosic (NFC) hydrogel as the scaffold. Light microscopic observations and F-actin staining confirmed the formation of spheroids. The proliferation efficiency indicated an expansion of cell population and an increase in spheroid size over time. The distribution, interaction pattern and influence of fibroblasts on the epithelial cell types were observed in terms of the size and shape of the HTS against homo-spheroids. An interesting observation was that, with an increase in the size of HTSs, many more fibroblast cells were found to occupy the core region, which, perhaps, was due to the faster growth of tumor cells over normal cells. Thus, normal and tumor cells, especially with origins from two different species, can be cultured together in 3D format, and this can potentially enhance our knowledge of tumor microenvironments and cell-cell interaction. These spheroids could be used to improve microphysiological systems for drug discovery and to better understand the tumor microenvironment.

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.

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease.

Advanced 3D in vitro models are laborious to prepare and susceptible to unintentional design errors due to culture adaptations, cell immaturity, xenofactors or yet incomplete knowledge of the dynamics within tissues or materials. In order to acquire cost-efficient research material with intact in vivo composition, we developed novel tissue culture method with plant-derived scaffolding. Human skin-, foreskin- and glioblastoma multiforme biopsies were dissected mechanically and cultivated for 28 days in plant-derived nanofibrillar cellulose hydrogel. Comparative cultures were done using mouse sarcoma tumor –derived Matrigel™. Long-term preservation of cultivated tissues was evaluated against typical immunohistochemical biomarkers for each tissue type: skin tissues for cytokeratins 5/6, E-cadherin and vimentin for sustained tissue structures, and brain neoplasia for Olig2, S100, Nestin, NOTCH1, MAP2 and GFAP for preserved disease profile. Histological analysis from both culture conditions showed that until day 28, all cultivated biopsy types were able to sustain their characteristic protein expressions without signs of necrosis. We here conclude a novel tissue culture model in xeno-free 3D scaffolding, that can enable long-term sample storage in vitro, studies of human tumor tissues and their non-neoplastic microenvironment, and innovations in personalized medicine research.

Background Ex vivo drug screening refers to the out-of-body assessment of drug efficacy in patient derived vital tumor cells. The purpose of these methods is to enable functional testing of patient specific efficacy of anti-cancer therapeutics and personalized treatment strategies. Such approaches could prove powerful especially in context of rare cancers for which demonstration of novel therapies is difficult due to the low numbers of patients. Here, we report comparison of different ex vivo drug screening methods in a metastatic urachal adenocarcinoma, a rare and aggressive non-urothelial bladder malignancy that arises from the remnant embryologic urachus in adults. Methods To compare the feasibility and results obtained with alternative ex vivo drug screening techniques, we used three different approaches; enzymatic cell viability assay of 2D cell cultures and image-based cytometry of 2D and 3D cell cultures in parallel. Vital tumor cells isolated from a biopsy obtained in context of a surgical debulking procedure were used for screening of 1160 drugs with the aim to evaluate patterns of efficacy in the urachal cancer cells. Results Dose response data from the enzymatic cell viability assay and the image-based assay of 2D cell cultures showed the best consistency. With 3D cell culture conditions, the proliferation rate of the tumor cells was slower and potency of several drugs was reduced even following growth rate normalization of the responses. MEK, mTOR, and MET inhibitors were identified as the most cytotoxic targeted drugs. Secondary validation analyses confirmed the efficacy of these drugs also with the new human urachal adenocarcinoma cell line (MISB18) established from the patient’s tumor. Conclusions All the tested ex vivo drug screening methods captured the patient’s tumor cells’ sensitivity to drugs that could be associated with the oncogenic KRAS G12V mutation found in the patient’s tumor cells. Specific drug classes however resulted in differential dose response profiles dependent on the used cell culture method indicating that the choice of assay could bias results from ex vivo drug screening assays for selected drug classes.

Abstract We have set up a precision medicine strategy for solid tumors to i) understand biological heterogeneity and driver signaling pathways in cancer, ii) identify new drug opportunities, iii) develop biomarkers for drug responses, and iv) eventually tailor effective treatments for individual patients. Fresh cancer tissue is obtained directly from clinics and processed to provide patient -derived cells (PDCs). PDCs and original tumor tissues are characterized using genetic profiling and image-based phenotyping, phenomics. Systematic drug sensitivity and resistance testing (DSRT) is carried out on the representative PDC models. The PDCs are plated in 384-well plates and treated with oncological compounds, each in five concentrations in 384-well plates. The cell viability and toxicity are measured as drug responses using plate readers. Alternatively, drug-treated cells are immunostained and subjected to automated high-content imaging and image analysis. Central to the success of this approach is to grow and test the patient derived cells in ex vivo conditions mimicking the tumor microenvironment. To this aim, we have developed 3D drug profiling for PDC spheroids from ovarian and renal cancers. For the 3D drug profiling, cells were cultivated either in Matrigel or in cellulose-based hydrogel, GrowDex, which has earlier been shown to support 3D growth of cell lines and stem cells. The pipetting robot Biomek FXp (Beckman Coulter) was utilized for transfer of matrices and cells to 384-well plates, and acoustic dispenser Echo 550 (Labcyte) for delivery of tailored drug library of 52 drugs to the spheroids. The drug sensitivities of cancer cell spheroids were scored using cell viability measurement as well as high-content confocal imaging. Our results with primary cells and cell lines suggest that both Matrigel and cellulose-based hydrogel are applicable in the 384-well plate drug profiling assay, and PDC spheroids are formed in both conditions. When the drug responses of ovarian cancer PDCs grown in different 2D and 3D conditions were systematically compared, we observed significant differences in sensitivity to several drugs. Here, we describe the individual drug effects in all conditions, such as some chemotherapeutics being less effective in 3D. As a conclusion, the comparison of results from 2D and 3D drug profiling increases our understanding of mechanisms of drugs, and may aid to select the most representative drugs for the patient. Citation Format: Piia Mikkonen, Laura Turunen, Lauri Paasonen, Swapnil Potdar, Lassi Paavolainen, Astrid Murumägi, Olli Kallioniemi, Vilja M. Pietiäinen. Precision cancer medicine based on 3D drug profiling of patient-derived cancer cell spheroid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5029.

Cell surface p32, the target of LyP-1 homing peptide, is upregulated in tumors and atherosclerotic plaques and has been widely used as a receptor for systemic delivery of payloads. Here, we identified an improved LyP-1-mimicking peptide (TT1, CKRGARSTC). We used this peptide in a fluorescence polarization-based high-throughput screening of a 50,000-compound chemical library and identified a panel of compounds that bind p32 with low micromolar affinity. Among the hits identified in the screen, two compounds were shown to specifically bind to p32 in multiple assays. One of these compounds was chosen for an in vivo study. Nanoparticles surface-functionalized with this compound specifically adhered to surfaces coated with recombinant p32 and, when injected intravenously, homed to p32-expressing breast tumors in mice. This compound provides a lead for the development of p32-targeted affinity ligands that circumvent some of the limitations of peptide-based probes in guided drug delivery.

Liposomes embedded with gold nanoparticles show light-triggered contents release. We investigated the mechanism of the light-induced changes and functionality of the light-induced release in the cells. The real time small angle X-ray scattering (SAXS) analysis revealed time-dependent phase transitions in distearoylphosphatidylcholine (DSPC)/dipalmitoylphosphatidylcholine (DPPC) liposomes upon heating. Similar changes were observed when gold nanoparticle-embedded liposomes were exposed to the UV light: gold nanoparticles absorb light energy and transfer it to heat, thereby causing lipid phase transition from gel phase to rippled phase, and further to fluid phase. Without UV light exposure the gold nanoparticles did not affect the liposomal bilayer periodicity. The light-triggered release of hydrophilic fluorescent probe (calcein) from the gold nanoparticle-loaded liposomes was demonstrated with fluorescence-activated cell sorting after liposome internalization into the ARPE-19 cells. The liposome formulations did not decrease the cell viability in vitro. In conclusion, the light-triggered release from the liposomes is functional in the cells, and the release is triggered by thermal phase changes in the lipid bilayers.

In the present study, synchrotron small-angle X-ray scattering (SAXS) combined with a UV light source (in situ SAXS-UV irradiation) was used to determine the structure response of gold nanoparticle (NP)-loaded. vesicles. The investigated system consisted of multilamellar vesicles of N-methylated dioleoylphosphatidylethanolamine (DOPE-Me) containing hydrophilic gold NPs with a size of 40 angstrom. Our results indicate drastic optothermically induced structural changes in these gold NP-loaded aqueous dispersions, which are accompanied. by strong alterations in the phospholipid membrane permeability. A structural mechanism from well-ordered multilamellar vesicles in the fluid lamellar (L(alpha)) phase to an inverted type hexagonal liquid crystalline (H(2)) phase via the formation of an intermediate phase of uncorrelated fluid bilayers is proposed. In general, these investigations are also crucial for the understanding of the potential of gold NP-loaded model phospholipid systems as efficient drug nanocarriers aiming to improve drug targeting and releasing on demand.

A novel proof of principle demonstration for contents release from liposomes that can be selectively activated by light irradiation is presented. The content release temperature was adjusted to slightly above body temperature, and hydrophobic or hydrophilic gold nanoparticles were incorporated into the lipid bilayer or the core of the liposomes, respectively. The release of a fluorescent marker was monitored upon exposure of the liposomes to UV light. Gold nanoparticle-containing liposomes remained intact at 37 C but contents release was triggered by UV light-induced heating of the gold nanoparticles. This light-induced release is mediated by heat transfer from the gold nanoparticles to the lipids and subsequent phase transition. Heating is highly localized in the liposomes and the gold nanoparticles act as energy collectors that sensitize the liposomes to the light signal. This kind of selectivity is very advantageous as it can potentially make the drug delivery mechanism biologically more compatible. The triggered contents release could also be extended to other applications where local contents release is needed. (c) 2007 Elsevier B.V. All rights reserved.