Development of human cell based in vitro vascular and cardiovascular models

Ihmissolupohjaisen verisuoni- ja sydänmallin kehittäminen Lääkkeiden ja teollisuuskemikaalien turvallisuuden arviointi on säädetty EU:ssa pakolliseksi ihmisten terveyden ja ympäristönsuojelun takaamiseksi. Uusien lääkeaineiden kehittämisessä suurin ongelma on niiden riittämätön teho tai lääkeaineiden ihmisille aiheuttamat haittavaikutukset. Tähän on syynä usein se, että eläinmalleilla ei ole kyetty ennustamaan lääkeaineiden ja kemikaalien tehoa tai turvallisuutta ihmisille. Huonon ennustavuuden lisäksi eettiset syyt ja korkeat kustannukset ovat syitä sille, että eläinbiologiaan perustuvat testausmallit halutaan korvata kehittyneemmillä menetelmillä. Ihmissolupohjaisten solu- ja kudosmallien avulla on mahdollista saada paremmin ihmiseen soveltuvaa tutkimustietoa. Ihmisen erittäin monikykyisistä kantasoluista erilaistetut solut tarjoavat mahdollisuuden yksilöllisten solumallien kehittämiseen lääkeaineiden ja kemikaalien turvallisuuden ja tehon testausta varten. Viimeaikaiset tutkimukset ovat osoittaneet, että yksisoluviljelmien sijaan muut oleelliset solutyypit ja kasvuympäristö ovat kriittisiä uusien, ennustavampien solumallien kehityksessä. Jotta solumallit voidaan hyväksyä virallisiksi testimenetelmiksi on tärkeää varmistaa, että mallit on huolellisesti kartoitettu ja että ne täyttävät niille asetetut vaatimukset ja sopivat aiottuun käyttötarkoitukseensa (validointi). Väitöskirjatyössä kehitettiin ihmissolupohjainen verisuoni- ja sydänmalli testimenetelmiksi, joilla voitaisiin täydentää tai korvata eläinkokeita lääkeaineiden ja kemikaalien tehon ja turvallisuuden tutkimisessa sekä käyttää malleja biolääketieteellisessä perustutkimuksessa. Työssä kartoitettiin verisuoni- ja sydänmallien toiminnallisuutta, rakennetta ja geneettisiä ominaisuuksia. Tulokset osoittivat, että ihmisen rasvakudoksen kantasolujen sekä endoteelisolujen muodostamassa yhteisviljelmässä muodostuu toistettavasti kypsiä verisuonimaisia rakenteita. Verisuonimallin kypsymistä aikuisen verisuoniston kaltaiseksi tuki työssä kehitetty uusi kasvatusliuos ja mallin todettiin olevan valmis laboratorion sisäiseen validointiin. Sydänmallissa yhdistettiin em. verisuonimaiset rakenteet sykkiviin sydänlihassoluihin. Tulokset osoittivat, että verisuonimaiset rakenteet edesauttavat sydänlihassolujen elävyyttä ja toiminnallisuutta. Täysin ihmissolupohjaisen sydänmallin osoitettiin vastaavan kemikaalialtistuksiin yksisoluviljelmää herkemmin ja omaavan aikuisen sydänlihaskudoksen kaltaisia rakenteellisia ominaisuuksia. Väitöskirjatyön tulosten perusteella verisuoni- ja sydänmalli tarjoavat yksisoluviljelmiä kehittyneemmän testausmenetelmän yhdisteiden turvallisuuden ja tehon testaamiseen sekä mahdollisuuden korvata osa vastaavista eläinmalleilla suoritettavista testeistä. Sydänmalli vaatii kuitenkin jatkokartoitusta sekä vertailua aikuisen ihmisen sydänlihaskudokseen ennen siirtymistä mallin validointiin.In order to protect human health and environment, safety assessment of drugs and industrial chemicals is mandatory according to the EU legislations. In pharmaceutical industry, lack of efficacy in addition to safety concerns in clinical trials are the main reasons for low success rate in the development of new drugs. Animal biology based test systems have often failed to predict the efficacy in humans and to reveal the adverse effects. In addition to poor predictive value, ethical concerns and high costs have raised the need to replace animal models and to develop more advanced test systems. Primary cells are considered as the traditional in vitro test systems for safety and efficacy assessment. More recently, human pluripotent stem cells have emerged as a promising source of specific cell types with the possibility for high throughput production with reasonably low costs. Growing data shows that instead of planar monocultures, supportive microenvironment, essential cell types and defined culture conditions are critical in developing more accurate in vitro models. Furthermore, before utilization in regulatory safety and efficacy assessment, careful characterization and validation of the developed in vitro models is necessary. The main objective of this thesis was to develop advanced, human cell based tissue models to supplement or, preferably, replace animal tests and to be used in biomedical research. First, in vitro vascular models were developed for toxicity and efficacy assessment of pro- or anti- angiogenic compounds. Careful characterization with defined medium was performed to vasculogenesis-angiogenesis model for further intra-laboratory validation and to study the properties of the model as a supportive platform for tissue models. As the second main objective, we combined the vascular model with cardiomyocytes to establish a cardiovascular model for cardiac safety and efficacy assessment of chemicals. The results showed that an extensive vascular-like network formation with mature tubules was reproducible formed in vasculogenesis-angiogenesis model in six day culture. The characterization with defined, serum-free medium showed that vasculogenesis-angiogenesis model is ready for intra-laboratory validation. Proof-of-concept on the enhanced viability of co-culture of cardiomyocytes and vascular-like network was received with two different vascular platforms. Finally, completely human cell based cardiovascular model was shown to possess more mature structure and response to chemicals than widely used cardiomyocyte monoculture. It can be concluded that the developed vascular and cardiovascular models provide more advanced test systems for safety and efficacy assessment compared to widely used monocultures with the possibility to supplement or replace part of the tests currently performed with animal models. However, further characterization as well as in vitro-in vivo comparison on human cardiovascular model is needed before it may enter into validation

Bioactivated gellan gum hydrogels affect cellular rearrangement and cell response in vascular co-culture and subcutaneous implant models

Hydrogels are suitable soft tissue mimics and capable of creating pre-vascularized tissues, that are useful for in vitro tissue engineering and in vivo regenerative medicine. The polysaccharide gellan gum (GG) offers an intriguing matrix material but requires bioactivation in order to support cell attachment and transfer of biomechanical cues. Here, four versatile modifications were investigated: Purified NaGG; avidin-modified NaGG combined with biotinylated fibronectin (NaGG-avd); oxidized GG (GGox) covalently modified with carbohydrazide-modified gelatin (gelaCDH) or adipic hydrazide-modified gelatin (gelaADH). All materials were subjected to rheological analysis to assess their viscoelastic properties, using a time sweep for gelation analysis, and subsequent amplitude sweep of the formed hydrogels. The sweeps show that NaGG and NaGG-avd are rather brittle, while gelatin-based hydrogels are more elastic. The degradation of preformed hydrogels in cell culture medium was analyzed with an amplitude sweep and show that gelatin-containing hydrogels degrade more dramatically. A co-culture of GFP-tagged HUVEC and hASC was performed to induce vascular network formation in 3D for up to 14 days. Immunofluorescence staining of the αSMA+ network showed increased cell response to gelatin-GG networks, while the NaGG-based hydrogels did not allow for the elongation of cells. Preformed, 3D hydrogels disks were implanted to subcutaneous rat skin pockets to evaluate biological in vivo response. As visible from the hematoxylin and eosin-stained tissue slices, all materials are biocompatible, however gelatin-GG hydrogels produced a stronger host response. This work indicates, that besides the biochemical cues added to the GG hydrogels, also their viscoelasticity greatly influences the biological response.publishedVersionPeer reviewe

Co-culture of human induced pluripotent stem cell-derived retinal pigment epithelial cells and endothelial cells on double collagen-coated honeycomb films

In vitro cell culture models representing the physiological and pathological features of the outer retina are urgently needed. Artificial tissue replacements for patients suffering from degenerative retinal diseases are similarly in great demand. Here, we developed a co-culture system based solely on the use of human induced pluripotent stem cell (hiPSC)-derived cells. For the first time, hiPSC-derived retinal pigment epithelium (RPE) and endothelial cells (EC) were cultured on opposite sides of porous polylactide substrates prepared by breath figures (BF), where both surfaces had been collagen-coated by Langmuir–Schaefer (LS) technology. Small modifications of casting conditions during material preparation allowed the production of free-standing materials with distinct porosity, wettability and ion diffusion capacity. Complete pore coverage was achieved by the collagen coating procedure, resulting in a detectable nanoscale topography. Primary retinal endothelial cells (ACBRI181) and umbilical cord vein endothelial cells (hUVEC) were utilised as EC references. Mono-cultures of all ECs were prepared for comparison. All tested materials supported cell attachment and growth. In mono-culture, properties of the materials had a major effect on the growth of all ECs. In co-culture, the presence of hiPSC-RPE affected the primary ECs more significantly than hiPSC-EC. In consistency, hiPSC-RPE were also less affected by hiPSC-EC than by the primary ECs. Finally, our results show that the modulation of the porosity of the materials can promote or prevent EC migration. In short, we showed that the behaviour of the cells is highly dependent on the three main variables of the study: the presence of a second cell type in co-culture, the source of endothelial cells and the biomaterial properties. The combination of BF and LS methodologies is a powerful strategy to develop thin but stable materials enabling cell growth and modulation of cell-cell contact. Statement of significance: Artificial blood-retinal barriers (BRB), mimicking the interface at the back of the eye, are urgently needed as physiological and disease models, and for tissue transplantation targeting patients suffering from degenerative retinal diseases. Here, we developed a new co-culture model based on thin, biodegradable porous films, coated on both sides with collagen, one of the main components of the natural BRB, and cultivated endothelial and retinal pigment epithelial cells on opposite sides of the films, forming a three-layer structure. Importantly, our hiPSC-EC and hiPSC-RPE co-culture model is the first to exclusively use human induced pluripotent stem cells as cell source, which have been widely regarded as an practical candidate for therapeutic applications in regenerative medicine.publishedVersionPeer reviewe

Building blocks of microphysiological system to model physiology and pathophysiology of human heart

Microphysiological systems (MPS) are drawing increasing interest from academia and from biomedical industry due to their improved capability to capture human physiology. MPS offer an advanced in vitro platform that can be used to study human organ and tissue level functions in health and in diseased states more accurately than traditional single cell cultures or even animal models. Key features in MPS include microenvironmental control and monitoring as well as high biological complexity of the target tissue. To reach these qualities, cross-disciplinary collaboration from multiple fields of science is required to build MPS. Here, we review different areas of expertise and describe essential building blocks of heart MPS including relevant cardiac cell types, supporting matrix, mechanical stimulation, functional measurements, and computational modelling. The review presents current methods in cardiac MPS and provides insights for future MPS development with improved recapitulation of human physiology.Peer reviewe

Perehdytysmateriaalien päivittäminen HSOY ry:lle

Tämä opinnäytetyö toteutettiin toimeksiantona Haaga-Helian ainejärjestö HSOY ry:lle. Toiminnallisen opinnäytetyön tavoitteena oli päivittää perehdytysmateriaalit ajantasaisiksi, huomioiden HSOY ry:ssä vuosien saatossa tapahtuneet muutokset. Ajantasainen perehdytysmateriaali palvelee niin perehdyttäjiä kuin perehdytettäviä yhdistyksessä. Opinnäytetyön tietoperustassa käsitellään yhdistystoimintaa ja perehdytystä. Toiminnallinen opinnäyte työ koostuu kahdesta osasta: tuotoksesta sekä opinnäytetyöraportista. Opinnäytetyön pohjalta syntyi päivitetyt perehdytysmateriaalit ja muistilista perehdytyksen tueksi. Perehdytysmateriaalit ovat HSOY ry:n hallituksen löydettävissä sähköisesti ja sitä tullaan käyttämään perehdytyksen tukena hallituskausien vaihtuessa. Perehdytysmateriaalit ovat tehty syksyn 2022 ja kevään 2023 aikana

Development of human cell based in vitro vascular and cardiovascular models

Ihmissolupohjaisen verisuoni- ja sydänmallin kehittäminen Lääkkeiden ja teollisuuskemikaalien turvallisuuden arviointi on säädetty EU:ssa pakolliseksi ihmisten terveyden ja ympäristönsuojelun takaamiseksi. Uusien lääkeaineiden kehittämisessä suurin ongelma on niiden riittämätön teho tai lääkeaineiden ihmisille aiheuttamat haittavaikutukset. Tähän on syynä usein se, että eläinmalleilla ei ole kyetty ennustamaan lääkeaineiden ja kemikaalien tehoa tai turvallisuutta ihmisille. Huonon ennustavuuden lisäksi eettiset syyt ja korkeat kustannukset ovat syitä sille, että eläinbiologiaan perustuvat testausmallit halutaan korvata kehittyneemmillä menetelmillä. Ihmissolupohjaisten solu- ja kudosmallien avulla on mahdollista saada paremmin ihmiseen soveltuvaa tutkimustietoa. Ihmisen erittäin monikykyisistä kantasoluista erilaistetut solut tarjoavat mahdollisuuden yksilöllisten solumallien kehittämiseen lääkeaineiden ja kemikaalien turvallisuuden ja tehon testausta varten. Viimeaikaiset tutkimukset ovat osoittaneet, että yksisoluviljelmien sijaan muut oleelliset solutyypit ja kasvuympäristö ovat kriittisiä uusien, ennustavampien solumallien kehityksessä. Jotta solumallit voidaan hyväksyä virallisiksi testimenetelmiksi on tärkeää varmistaa, että mallit on huolellisesti kartoitettu ja että ne täyttävät niille asetetut vaatimukset ja sopivat aiottuun käyttötarkoitukseensa (validointi). Väitöskirjatyössä kehitettiin ihmissolupohjainen verisuoni- ja sydänmalli testimenetelmiksi, joilla voitaisiin täydentää tai korvata eläinkokeita lääkeaineiden ja kemikaalien tehon ja turvallisuuden tutkimisessa sekä käyttää malleja biolääketieteellisessä perustutkimuksessa. Työssä kartoitettiin verisuoni- ja sydänmallien toiminnallisuutta, rakennetta ja geneettisiä ominaisuuksia. Tulokset osoittivat, että ihmisen rasvakudoksen kantasolujen sekä endoteelisolujen muodostamassa yhteisviljelmässä muodostuu toistettavasti kypsiä verisuonimaisia rakenteita. Verisuonimallin kypsymistä aikuisen verisuoniston kaltaiseksi tuki työssä kehitetty uusi kasvatusliuos ja mallin todettiin olevan valmis laboratorion sisäiseen validointiin. Sydänmallissa yhdistettiin em. verisuonimaiset rakenteet sykkiviin sydänlihassoluihin. Tulokset osoittivat, että verisuonimaiset rakenteet edesauttavat sydänlihassolujen elävyyttä ja toiminnallisuutta. Täysin ihmissolupohjaisen sydänmallin osoitettiin vastaavan kemikaalialtistuksiin yksisoluviljelmää herkemmin ja omaavan aikuisen sydänlihaskudoksen kaltaisia rakenteellisia ominaisuuksia. Väitöskirjatyön tulosten perusteella verisuoni- ja sydänmalli tarjoavat yksisoluviljelmiä kehittyneemmän testausmenetelmän yhdisteiden turvallisuuden ja tehon testaamiseen sekä mahdollisuuden korvata osa vastaavista eläinmalleilla suoritettavista testeistä. Sydänmalli vaatii kuitenkin jatkokartoitusta sekä vertailua aikuisen ihmisen sydänlihaskudokseen ennen siirtymistä mallin validointiin.In order to protect human health and environment, safety assessment of drugs and industrial chemicals is mandatory according to the EU legislations. In pharmaceutical industry, lack of efficacy in addition to safety concerns in clinical trials are the main reasons for low success rate in the development of new drugs. Animal biology based test systems have often failed to predict the efficacy in humans and to reveal the adverse effects. In addition to poor predictive value, ethical concerns and high costs have raised the need to replace animal models and to develop more advanced test systems. Primary cells are considered as the traditional in vitro test systems for safety and efficacy assessment. More recently, human pluripotent stem cells have emerged as a promising source of specific cell types with the possibility for high throughput production with reasonably low costs. Growing data shows that instead of planar monocultures, supportive microenvironment, essential cell types and defined culture conditions are critical in developing more accurate in vitro models. Furthermore, before utilization in regulatory safety and efficacy assessment, careful characterization and validation of the developed in vitro models is necessary. The main objective of this thesis was to develop advanced, human cell based tissue models to supplement or, preferably, replace animal tests and to be used in biomedical research. First, in vitro vascular models were developed for toxicity and efficacy assessment of pro- or anti- angiogenic compounds. Careful characterization with defined medium was performed to vasculogenesis-angiogenesis model for further intra-laboratory validation and to study the properties of the model as a supportive platform for tissue models. As the second main objective, we combined the vascular model with cardiomyocytes to establish a cardiovascular model for cardiac safety and efficacy assessment of chemicals. The results showed that an extensive vascular-like network formation with mature tubules was reproducible formed in vasculogenesis-angiogenesis model in six day culture. The characterization with defined, serum-free medium showed that vasculogenesis-angiogenesis model is ready for intra-laboratory validation. Proof-of-concept on the enhanced viability of co-culture of cardiomyocytes and vascular-like network was received with two different vascular platforms. Finally, completely human cell based cardiovascular model was shown to possess more mature structure and response to chemicals than widely used cardiomyocyte monoculture. It can be concluded that the developed vascular and cardiovascular models provide more advanced test systems for safety and efficacy assessment compared to widely used monocultures with the possibility to supplement or replace part of the tests currently performed with animal models. However, further characterization as well as in vitro-in vivo comparison on human cardiovascular model is needed before it may enter into validation

Human vascular model with defined stimulation medium - a characterization study

The formation of blood vessels is a vital process in embryonic development and in normal physiology. Current vascular modelling is mainly based on animal biology leading to species-to-species variation when extrapolating the results to humans. Although there are a few human cell based vascular models available, these assays are insufficiently characterized in terms of culture conditions and developmental stage of vascular structures. Therefore, well characterized vascular models with human relevance are needed for basic research, embryotoxicity testing, development of therapeutic strategies and for tissue engineering. We have previously shown that the in vitro vascular model based on co-culture of human adipose stromal cells (hASC) and human umbilical vein endothelial cells (HUVEC) is able to induce an extensive vascular-like network with high reproducibility. In this work we developed a defined serum-free vascular stimulation medium (VSM) and performed further characterization in terms of cell identity, maturation and structure to obtain a thoroughly characterized in vitro vascular model to replace or reduce corresponding animal experiments. The results showed that the novel vascular stimulation medium induced an intact and evenly distributed vascular-like network with morphology of mature vessels. Electron microscopic analysis assured the three-dimensional microstructure of the network containing lumen. Additionally, elevated expression levels of the main human angiogenesis-related genes were detected. In conclusion, with the newly defined medium the vascular model can be utilized as a characterized test system for chemical testing as well as in creating vascularized tissue models

Simultaneous induction of vasculature and neuronal network formation on a chip reveals a dynamic interrelationship between cell types

Background: Neuronal networks receive and deliver information to regulate bodily functions while the vascular network provides oxygen, nutrients, and signaling molecules to tissues. Neurovascular interactions are vital for both tissue development and maintaining homeostasis in adulthood; these two network systems align and reciprocally communicate with one another. Although communication between network systems has been acknowledged, the lack of relevant in vitro models has hindered research at the mechanistic level. For example, the current used in vitro neurovascular models are typically established to be short-term (≤ 7 days) culture models, and they miss the supporting vascular mural cells. Methods: In this study, we utilized human induced pluripotent stem cell (hiPSC) -derived neurons, fluorescence tagged human umbilical vein endothelial cells (HUVECs), and either human bone marrow or adipose stem/stromal cells (BMSCs or ASCs) as the mural cell types to create a novel 3D neurovascular network-on-a-chip model. Collagen 1–fibrin matrix was used to establish long-term (≥ 14 days) 3D cell culture in a perfusable microphysiological environment. Results: Aprotinin-supplemented endothelial cell growth medium-2 (EGM-2) supported the simultaneous formation of neuronal networks, vascular structures, mural cell differentiation, and the stability of the 3D matrix. The formed neuronal and vascular networks were morphologically and functionally characterized. Neuronal networks supported vasculature formation based on direct cell contacts and by dramatically increasing the secretion of angiogenesis-related factors in multicultures in contrast to cocultures without neurons. Both utilized mural cell types supported the formation of neurovascular networks; however, the BMSCs seemed to boost neurovascular networks to greater extent. Conclusions: Overall, our study provides a novel human neurovascular network model that is applicable for creating in vivo-like tissue models with intrinsic neurovascular interactions. The 3D neurovascular network model on chip forms an initial platform for the development of vascularized and innervated organ-on-chip and further body-on-chip concepts and offers the possibility for mechanistic studies on neurovascular communication both under healthy and in disease conditions. [MediaObject not available: see fulltext.]Peer reviewe