In vitro alveolar-capillary models based on co-culturing the alveolar epithelial cell line H441 and primary pulmonary microvascular endothelial cells (HPMEC) are a widely used platform for evaluating the function of the alveolar barrier. However, the relatively thick synthetic membranes that are used as substrates in most approaches fail to mimic the properties of the natural basement membrane, thereby decreasing the physiological relevance of those models. We investigated the potential of the FN-silk membrane to support an in vitro alveolar-capillary model. The FN-silk membrane is micrometer-thin, fibrillar, constructed from a functionalized recombinant spider silk protein, and has been previously shown to be a potent basement membrane mimic supporting physiologically relevant in vitro models of various barrier tissues (i.e., blood vessel, skin, BBB, and kidney). Herein, it supported alveolar epithelial and endothelial barrier formation, surfactant protein B and C (SPB, SPC) production, and epithelial cell polarization, detected with immunofluorescence. Notably, we also demonstrated for the first time, to our knowledge, that key events related to alveologenesis (i.e., cell hollowing, lumen formation, septation, and α-SMA expression) can take place in an in vitro model. This further highlights the ability of the FN-silk membrane to recapitulate the complicated alveolar milieu and expanding the known potential of the H441 cell line, which to our knowledge, has not been previously reported to enable modeling the alveolar tissue 3D morphology. We propose the FN-silk-based alveolar-capillary model as an advanced in vitro model that can be used as a potent tool in respiratory, developmental biology, and regenerative medicine research.

Spider silk is a natural protein-based material known for its medicinal use and remarkable mechanical properties. Structures made thereof are both strong and elastic and have been shown to be favorable matrices for tissue engineering. As natural spider silk is difficult to obtain, recombinant technology is instead used to produce partial silk proteins. This thesis investigates the use of one such partial spider silk protein functionalized with a cell adhesion motif from fibronectin, FN-4RepCT, to coat the surface of synthetic polymers, as well as self-assemble into nanofibrillar membranes for modelling of tissues in vitro.

In Paper I, the silk protein was shown to self-assemble into coatings with simultaneous entrapment of cells (co-seeding) to functionalize polymeric surfaces. The results showed that the co-seeding approach facilitated the adherence and sustained the viability of cells on surfaces of materials widely used for the manufacture of cardiovascular grafts.

In Paper II, a protocol was developed to enable the formation of nanofibrillar coatings on the surface of membranes intended for guided bone regeneration. This was done by reducing the surface tension of the membranes, allowing for the self-assembly of silk proteins to take place. The silk coating facilitated the adherence, promoted the growth, and mediated the generation of a cell monolayer of tissue representative cells seeded on either side of the membrane.

In Paper III, the self-assembly of the silk protein at the air-liquid interface was shown to form cm-sized free-standing, tough and elastic, nanofibrillar silk nanomembranes, permeable to macromolecules of various sizes, and able to support the establishment of a confluent layer of keratinocytes seeded on either side. In Paper IV, the nanofibrillar silk membranes were shown able to support cell co-culture to generate a model of the blood vessel wall in vitro.

In Paper V, an alveolar-capillary model was established by seeding lung epithelial and endothelial cells on opposite sides of nanofibrillar silk membranes. The results showed the formation of an in vivo like tissue through the expression of junctional complexes and the production of essential surfactants. The silk membranes were also for the first time integrated into a microfluidic device to expose the endothelium to flow-induced shear stresses.

Altogether, the work conducted in this thesis shows promise to the use of the FN-4RepCT silk protein both for coating surfaces of bio-inert synthetic polymeric materials and forming thin and nanofibrillar membranes for the engineering of tissues in vitro.

Spindelsilke är ett naturligt proteinbaserat material som är välkänt för sin medicinska användning och sina anmärkningsvärda mekaniska egenskaper. Strukturer gjorda därav är både starka och elastiska och har visat sig vara lämpliga matriser för vävnadsteknik. Eftersom det naturliga spindelsilket är svårt att få tag på, används i stället rekombinationsteknologi för att producera partiella silkesproteiner. Denna avhandling undersöker användningen av ett sådant partiellt spindelsilkeprotein som har funktionaliserats med ett celladhesionsmotiv från fibronektin, FN-4RepCT, för att belägga ytor på syntetiska polymerer, samt forma nanofibrillära membran för modellering av vävnader in vitro.

I Artikel I visades att silkesproteinet, tillsammans med celler, spontant formar en beläggning på ytan av olika polymerer, och därmed kan användas för att funktionalisera dessa. Resultaten visade att samformuleringsmetoden underlättar eller t.o.m. möjliggör adhesion av cellerna till ytan, samt bibehåller cellernas viabilitet på material som vanligen används i kardiovaskulära transplantat.

I Artikel II utvecklades ett protokoll för att minska ytspänningen hos membran som är avsedda för att styra benregenerering. Detta för att möjliggöra för silkesproteiner att forma nanofibrillära silkesbeläggningar på denna typ av material. Silkesbeläggningen underlättade adhesion av cellerna, och befrämjade deras tillväxt så att monolager av vävnadsrepresentativa celler kunde bildas på vardera sidan av membranet.

I Artikel III visas det att silkesproteinet spontant bildar cm-stora fristående, starka och elastiska silkesnanomembran vid gränssnittet mellan luft och vätska. Molekyler av olika storlekar kan passera genom membranen, som även främjar etableringen av ett konfluent monolager av keratinocyter sådda på båda sidorna av membranet.

I Artikel IV påvisas det att dessa silkesmembran kan användas för samodling av två celltyper och på så sätt skapa en in vitro modell av blodkärlsväggen.

I Artikel V etablerades en alveolär-kapillär in vitro modell genom att odla lungepitel- och endotelceller på motsatta sidor av nanofibrillära silkesmembran. Cellerna hade speciella strukturer på sin yta som förankrade dem med varandra, och producerade även essentiella yt-surfaktanter, vilket sammantaget visade att en in vivo-liknande vävnad hade bildats. Silkesmembranen integrerades också i ett mikroflödes-chip, något som aldrig tidigare genomförts, för att kunna exponera endotelet för flödesinducerade skjuvkrafter.

Sammantaget visar arbetet som utförts i denna avhandling att silkesproteinet FN-4RepCT är lovande för att belägga bioinerta ytor av syntetiska polymerer, samt för att bilda tunna, nanofibrillära membran för konstruktion av vävnader in vitro.

Self-assembly of recombinant spider silk protein at air-liquid interfaces is used as a starting point to produce homogeneous fiber bundles. The film that is formed on a silk protein solution in a vertically placed syringe is subjected to repeated controlled extension and compression by an oscillating vertical motion. Thereby, a precise breakup of the film can be achieved, followed by transport and roll-up against the syringe wall prior to extraction. Advantages of the method are that it 1) is simple to use; 2) requires a small volume of protein solution (1 mL) at relatively low concentration (1 mg mL^-1 ); 3) can be performed under sterile conditions; 4) does not require any use of coagulants; and 5) is compatible with the addition of viable cells during the process, which thereby are integrated uniformly throughout the fiber.

Basement membrane is a thin but dense network of self-assembled extracellular matrix (ECM) protein fibrils that anchors and physically separates epithelial/endothelial cells from the underlying connective tissue. Current replicas of the basement membrane utilize either synthetic or biological polymers but have not yet recapitulated its geometric and functional complexity highly enough to yield representative in vitro co-culture tissue models. In an attempt to model the vessel wall, we seeded endothelial and smooth muscle cells on either side of 470 +/- 110 nm thin, mechanically robust, and nanofibrillar membranes of recombinant spider silk protein. On the apical side, a confluent endothelium formed within 4 days, with the ability to regulate the permeation of representative molecules (3 and 10 kDa dextran and IgG). On the basolateral side, smooth muscle cells produced a thicker ECM with enhanced barrier properties compared to conventional tissue culture inserts. The membranes withstood 520 +/- 80 Pa pressure difference, which is of the same magnitude as capillary blood pressure in vivo. This use of protein nanomembranes with relevant properties for co-culture opens up for developing advanced in vitro tissue models for drug screening and potent substrates in organ-on-a-chip systems.

We show for the first time that 470 nm thick spider silk membranes support co-culturing of endothelial (HDMEC) and smooth muscle cells (SMC). These cell-silk-cell constructs mimic the wall of small blood vessels. The silk membranes are formed through self-assembly at the liquid:air interface of a standing solution of spider silk protein. We show that the silk membranes enable communication between the cells better than commercial tissue culture inserts (TC-inserts).

In this work we present three different ways to characterize the mechanical properties of spider silk nanomembranes. The nanomembranes are formed by self-assembly at the liquid:air interface of a standing solution from which they can be lifted. The mechanical properties are evaluated by (1) manually dropping lead bullets onto the nanomembrane, (2) motorized lowering of a cylindrical indenter to record force-deformation characteristics, and (3) using a standard bulging experiments. Using these methods we show that the nanomembranes are both strong and flexible opening up for applications as pneumatic actuators in MEMS microvalves, or as cell layer actuators in organ-on-a-chip. 

Biologically compatible membranes are of high interest for several biological and medical applications. Tissue engineering, for example, would greatly benefit from ultrathin, yet easy‐to‐handle, biodegradable membranes that are permeable to proteins and support cell growth. In this work, nanomembranes are formed by self‐assembly of a recombinant spider silk protein into a nanofibrillar network at the interface of a standing aqueous solution. The membranes are cm‐sized, free‐standing, bioactive and as thin as 250 nm. Despite their nanoscale thickness, the membranes feature an ultimate engineering strain of over 220% and a toughness of 5.2 MPa. Moreover, they are permeable to human blood plasma proteins and promote cell adherence and proliferation. Human keratinocytes seeded on either side of the membrane form a confluent monolayer within three days. The significance of these results lays in the unique combination of nanoscale thickness, elasticity, toughness, biodegradability, protein permeability and support for cell growth, as this may enable new applications in tissue engineering including bi‐layered in vitro tissue models and support for clinical transplantation of coherent cell layers.

Alveolar bone loss is usually treated with guided bone regeneration, a dental procedure which utilizes a tissue-separation membrane. The barrier membrane prevents pathogens and epithelial cells to invade the bone augmentation site, thereby permitting osteoblasts to deposit minerals and build up bone. This study aims at adding bioactive properties to otherwise inert PTFE membranes in order to enhance cell adherence and promote proliferation. A prewetting by ethanol and stepwise hydration protocol was herein employed to overcome high surface tension of PTFE membranes and allow for a recombinant spider silk protein, functionalized with a cell-binding motif from fibronectin (FN-silk), to self-assemble into a nanofibrillar coating. HaCaT and U-2 OS cells were seeded onto soft and hard tissue sides, respectively, of membranes coated with FN-silk. The cells could firmly adhere as early as 1 h post seeding, as well as markedly grow in numbers when kept in culture for 7 days. Fluorescence and scanning electron microscopy images revealed that adherent cells could form a confluent monolayer and develop essential cell–cell contacts during 1 week of culture. Hence, functionalized PTFE membranes have a potential of better integration at the implantation site, with reduced risk of membrane displacement as well as exposure to oral pathogens.

In vitro endothelialization of synthetic grafts or engineered vascular constructs is considered a promising alternative to overcome shortcomings in the availability of autologous vessels and in graft complications with synthetics. A number of cell-seeding techniques have been implemented to render vascular grafts accessible for cells to attach, proliferate, and spread over the surface area. Nonetheless, seeding efficiency and the time needed for cells to adhere varies dramatically. Herein, we investigated a novel cell-seeding approach (denoted co-seeding) that enables cells to bind to a motif from fibronectin included in a recombinant spider silk protein. Entrapment of cells occurs at the same time as the silk assembles into a nanofibrillar coating on various substrates. Cell adhesion analysis showed that the technique can markedly improve cell-seeding efficiency to nonfunctionalized polystyrene surfaces, as well as establish cell attachment and growth of human dermal microvascular endothelial cells on bare polyethylene terephthalate and polytetrafluoroethylene (PTFE) substrates. Scanning electron microscopy images revealed a uniform endothelial cell layer and cell-substratum compliance with the functionalized silk protein to PTFE surfaces. The co-seeding technique holds a great promise as a method to reliably and quickly cellularize engineered vascular constructs as well as to in vitro endothelialize commercially available cardiovascular grafts.