Markus Rimann

Synthetic 3D multicellular systems for drug development

Since the 1970s, the limitations of two dimensional (2D) cell culture and the relevance of appropriate three dimensional (3D) cell systems have become increasingly evident. Extensive effort has thus been made to move cells from a flat world to a 3D environment. While 3D cell culture technologies are meanwhile widely used in academia, 2D culture technologies are still entrenched in the (pharmaceutical) industry for most kind of cell-based efficacy and toxicology tests. However, 3D cell culture technologies will certainly become more applicable if biological relevance, reproducibility and high throughput can be assured at acceptable costs. Most recent innovations and developments clearly indicate that the transition from 2D to 3D cell culture for industrial purposes, for example, drug development is simply a question of time.

Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells.

Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application.

An in vitro osteosarcoma 3D microtissue model for drug development

Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Therapy today includes surgical removal of the tumour and neoadjuvant and adjuvant chemotherapy. The 5-year survival rates for patients with localised disease are between 50 and 70%, but in patients with metastases the prognosis remains poor (∼ 20%). The aim of this study was the development of a biological relevant OS 3D microtissue model, which is suitable for drug development. Microtissues were formed by the hanging drop method with the established OS cell lines SaOS-2, HOS and MG-63, as well as with cells derived from osteoblastic and chondroblastic OS patient material. Histological characterisation of the microtissues with H/E- and Ki-67-(proliferation), as well as apoptosis staining (TUNEL) revealed the inherent histological heterogeneity of OS. Microtissues from SaOS-2 and HOS cell lines were exposed to doxorubicin, cisplatin, taurolidine, pemetrexed and taxol and the viability was assessed by the CellTiter-GLO(®) Luminescent Cell Viability Assay. The obtained IC50-values for 3D cultures were all higher (1.7 to >16,000-fold) when compared to corresponding cells grown in 2D monolayer culture, except for pemetrexed that was inactive in 2D and 3D cultures. Doxorubicin did not affect the viability of chondroblastic monolayer cultures whereas on 3D microtissues an IC50-value of 2.3 μM was obtained. The 3D microtissues reflect the tissue heterogeneity of OS and are potential suitable tools for drug development towards personalised medicine.

Automation of 3D Cell Culture Using Chemically Defined Hydrogels

Drug development relies on high-throughput screening involving cell-based assays. Most of the assays are still based on cells grown in monolayer rather than in three-dimensional (3D) formats, although cells behave more in vivo–like in 3D. To exemplify the adoption of 3D techniques in drug development, this project investigated the automation of a hydrogel-based 3D cell culture system using a liquid-handling robot. The hydrogel technology used offers high flexibility of gel design due to a modular composition of a polymer network and bioactive components. The cell inert degradation of the gel at the end of the culture period guaranteed the harmless isolation of live cells for further downstream processing. Human colon carcinoma cells HCT-116 were encapsulated and grown in these dextran-based hydrogels, thereby forming 3D multicellular spheroids. Viability and DNA content of the cells were shown to be similar in automated and manually produced hydrogels. Furthermore, cell treatment with toxic Taxol concentrations (100 nM) had the same effect on HCT-116 cell viability in manually and automated hydrogel preparations. Finally, a fully automated dose-response curve with the reference compound Taxol showed the potential of this hydrogel-based 3D cell culture system in advanced drug development.

3D Bioprinted Muscle and Tendon Tissues for Drug Development.

IntroductionIn our aging societies, there is a huge medical need fortreatments of degenerative muscle and tendon diseases, forwhichtherearecurrentlynoapprovedpharmaceuticaltherapies.Furthermore, also devastating muscle diseases that affectchildren and younger patients such as Duchenne musculardystrophyoramyothrophiclateralsclerosis(ALS)lackcurativedrug treatments. A major hurdle in new drug discovery anddevelopment is the nonexistence of normal functional humantissuesanddiseasedtissuesforcompoundscreeningandtesting.Currently,mosthigh-throughputdrugscreeningcampaignsareperformedwithtarget-centeredbiochemicalorsimplehumancell-basedassaysifthedrugtargetisknown,orwithtwo-dimensional(2D)cellculturephenotypicscreens,ifthetargetisunknown.Identifiedhitsandfurtheroptimizedcompounds(leads)arethenusually analyzed in low-throughput rodent ex vivo and/or invivo preclinicalanimalmodelsforefficacy, potency, specificityandsafety.Besidestheobviousslownessofthissteptoassessthe pharmacodynamic and physiological effects of new drugcandidatesthejumpfromhumancell-basedsystemstoanimalpreclinicalmodelsandtohumanclinicaltrialsisveryoftentoolargeandnotreliableenoughtomaster.Fortunately,recentyearshaveseenanincredibleprogressinnewapproachesgeneratingfunctional3Dhumantissuesfromnormalanddiseasedonors.Three-dimensional(3D)humanorganotypictissueculturesaregenerallymorepredictiveforinvivoeffects,becausetheymodelmuchmorein vivo tissuephysiologythanconventional2Dcellcultures.Thus,3Dtissueculturehasthepotentialtorevolutionizedrugdiscoveryanddevelopment.Theparadigmshiftfrom2Dto3Dcellcultureisalreadyshowinggreatbenefitinbasicresearchof tissues differentiation and homeostasis as well as in tissueengineeringeffortsforregenerativemedicineapplications.

A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues

Two-dimensional (2D) cell cultures do not reflect the in vivo situation, and thus it is important to develop predictive three-dimensional (3D) in vitro models with enhanced reliability and robustness for drug screening applications. Treatments against muscle-related diseases are becoming more prominent due to the growth of the aging population worldwide. In this study, we describe a novel drug screening platform with automated production of 3D musculoskeletal-tendon-like tissues. With 3D bioprinting, alternating layers of photo-polymerized gelatin-methacryloyl-based bioink and cell suspension tissue models were produced in a dumbbell shape onto novel postholder cell culture inserts in 24-well plates. Monocultures of human primary skeletal muscle cells and rat tenocytes were printed around and between the posts. The cells showed high viability in culture and good tissue differentiation, based on marker gene and protein expressions. Different printing patterns of bioink and cells were explored and calcium signaling with Fluo4-loaded cells while electrically stimulated was shown. Finally, controlled co-printing of tenocytes and myoblasts around and between the posts, respectively, was demonstrated followed by co-culture and co-differentiation. This screening platform combining 3D bioprinting with a novel microplate represents a promising tool to address musculoskeletal diseases.

TEDD Annual Meeting with 3D Bioprinting Workshop

Bioprinting is the technology of choice for realizing functional tissues such as vascular system, muscle, cartilage and bone. In the future, bioprinting will influence the way we engineer tissues and bring it to a new level of physiological relevance. That was the topic of the 2017 TEDD Annual Meeting at ZHAW Waedenswil on 8th and 9th November. In an exciting workshop, the two companies regenHU Ltd. and CELLINK gave us an insight into highly topical applications and collaborations in this domain.

ZHAW Waedenswil: A new Approach in the Fight against Cancer

A happy coincidence brought Dr Markus Rimann from ZHAW Waedenswil together with Dr Andreas Meyer from the start-up FGen and PD Dr Emanuela Felley-Bosco, Molecular Oncologist at Zurich University Hospital, to develop a technology platform for the manufacture and high throughput analysis of single mesothelioma spheroids. Armin Picenoni, former student in Chemistry for the Life Sciences at ZHAW, confirmed everything in writing his Master Thesis on this Innosuisse project.

In vitro characterization of a new composite material for biomedical applications and 3D (bio)printing

biomedical applications and 3D (bio)printing Epifania Bono1, Christoph Evers2, Franca Schmid2, Ursula Graf-Hausner3, Markus Rimann1 1Zurich University of Applied Sciences ZHAW, Institute of Chemistry and Biotechnology ICBT, Einsiedlerstrasse 31, 8820 Waedenswil, Switzerland 2Saremco Dental AG, Gewerbestrasse 4, 9445 Rebstein, Switzerland 3graf3dcellculture, Bühlackerweg 5, 8405 Winterthur, Switzerland

Aktuelle Trends bei der in vitro-Substanztestung in Deutschland und der Schweiz

o Der Bedarf an Testsystemen fur chemische und pharmakologisch aktive Substanzen ist aufgrund regulatorischer Vorgaben (EU-Chemikalienverordnung REACH, Kosmetikverordnung) immens gestiegen. Anstelle der heute noch ublichen Tierversuche sollen kunftig organotypische Gewebekulturen treten, deren Aussagekraft eine hohere Relevanz verspricht. Tierversuche werfen zur ethischen Problematik weitere Limitierungen wie die unzureichende Verfugbarkeit oder die haufig nicht gewahrleistete Ubertragbarkeit von Daten aus dem Tiermodell auf den Menschen auf. Es wird erwartet, dass durch organotypische Gewebekulturen die Medikamentenentwicklung und Wirkstofftestung sicherer und vorhersehbarer werden. Die neuen Testverfahren sind des Weiteren durch die Verwendung von patientenspezifischem Material als Erganzung zu klinischen Studien zu sehen, da dadurch die genetische Vielfalt der Patienten besser berucksichtigt werden kann. Nach einer DECHEMA-Umfrage im Jahr 2009 zu akademischen und industriellen Forschungsaktivitaten auf dem Gebiet der Zellund Gewebekulturtechnik fur die regenerative Medizin und Substanzprufungen in Deutschland [1] wurde im Jahr 2013 eine Neubewertung der Aktivitaten in diesem Bereich durchgefuhrt und um die Aktivitaten in der Schweiz erweitert. Die Umfrage wurde zudem starker auf die in vitro-Substanztestung einschlieslich der Nutzung systembiologischer Methoden ausgerichtet. Auf deutscher Seite wurde die Studie durch die Beirate der Fachgruppen „Zellkulturtechnologie“ und „Medizinische Biotechnologie“ der DECHEMA e. V., auf schweizerischer Seite vom nationalen Kompetenzzentrum TEDD (Tissue Engineering for Drug Development and Substance Testing) getragen.