Towards More Predictive, Physiological and Animal-free In Vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings.

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.

Ex vivo modelling of drug efficacy in a rare metastatic urachal carcinoma.

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 KRASG12V 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.

New p32/gC1qR Ligands for Targeted Tumor Drug Delivery.

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.

Cellulase-assisted platelet-rich plasma release from nanofibrillated cellulose hydrogel enhances wound healing.

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.

Comparison of two supporting matrices for patient-derived cancer cells in 3D drug sensitivity and resistance testing assay (3D-DSRT).

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.

Rapid hydrogel-based phage susceptibility test for pathogenic bacteria.

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.

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.

Epidermal cell culture model with tight stratum corneum as a tool for dermal gene delivery studies.

The purpose of this study was to evaluate the feasibility of organotypic cultures of rat epidermal cells as a tool to study non-invasive dermal gene delivery. Also, a novel transfection method employing liposomal pre-treatment of stratum corneum (SC) was evaluated. Rat epidermal cells were cultured on Transwell tissue culture inserts and formation of stratum corneum barrier was evaluated in permeability studies with two model compounds. Transfections were performed with naked pCMV-SEAP2 plasmid and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/dioleyl-phosphatidylethanolamine (DOPE)/DNA lipoplexes. Naked DNA was administered on the stratum corneum of the cell culture model with or without prior treatment of the stratum corneum with DOTAP/DOPE liposomes. Transfection was evaluated non-invasively by monitoring concentrations of secreted alkaline phosphatase (SEAP) in the culture medium of the basolateral compartment at 24-h intervals. Transfection with lipoplexes produced significant gene expression in rat epidermal keratinocyte (REK) epidermal culture model. Likewise, delivery of naked DNA on stratum corneum after DOTAP/DOPE liposome pre-treatment produced gene expression. Naked DNA alone did not result in detectable gene expression. In dermal gene delivery studies REK epidermal culture model is a suitable tool that includes tight stratum corneum and allows transgene expression in viable epidermis and non-invasive sampling of secreted gene product in the basolateral compartment. Liposomal pre-treatment of the stratum corneum augments transfection of viable epidermis.

Gold-embedded photosensitive liposomes for drug delivery: triggering mechanism and intracellular release.

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.

Temperature-sensitive poly(N-(2-hydroxypropyl)methacrylamide mono/dilactate)-coated liposomes for triggered contents release.

We prepared thermosensitive poly( N-(2-hydroxypropyl)methacrylamide mono/dilactate) (pHPMA mono/dilactate) polymer and studied temperature-triggered contents release from polymer-coated liposomes. HPMA mono/dilactate polymer was synthesized with a cholesterol anchor suitable for incorporation in the liposomal bilayers and with a cloud point (CP) temperature of the polymer slightly above normal body temperature (42 degrees C). Dynamic light scattering (DLS) measurements showed that whereas the size of noncoated liposomes remained stable upon raising the temperature from 25 to 46 degrees C, polymer-coated liposomes aggregated around 43 degrees C. Also, noncoated liposomes loaded with calcein showed hardly any leakage of the fluorescent marker when heated to 46 degrees C. However, polymer-coated liposomes showed a high degree of temperature-triggered calcein release above the CP of the polymer. Likely, liposome aggregation and bilayer destabilization are triggered because of the precipitation of the thermosensitive polymer above its CP onto the liposomal bilayers, followed by permeabilization of the liposomal membrane. This study demonstrates that liposomes surface-modified with HPMA mono/dilactate copolymer are attractive systems for achieving temperature-triggered contents release.

Gold nanoparticles enable selective light-induced contents release from liposomes.

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 degrees 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.