Research Carina Koppen

Abstract

This innovation mandate aims to validate essential material properties of an innovative medical device that are crucial during surgery, both in the short and long term after the procedure. In this project, the focus is on basic research to further support the preliminary proof-of-concept. We have planned various qualification and validation experiments that the product must undergo, related to three phases: the procedure itself, short-term post-operation (the first four weeks), and long-term (months to years). Furthermore, market research is also conducted on two fronts. A roadmap is outlined for the preclinical development of the technology from the establishment of the spin-off, and a go-to-market strategy is being formulated. Using a SMART analysis, five parameters crucial to the functioning of the permanent contact lens were identified: shelf life, sterilization, laser impact, reversibility, and stability. This analysis forms the basis of the work packages of the basic research discussed in the project description.

Researcher(s)

• Promoter: Koppen Carina
• Fellow: Van den Bogerd Bert

Research team(s)

• Translational Neurosciences (TNW)

Project type(s)

• Research Project

Abstract

The corneal endothelium is the innermost layer of the human cornea, the eye's transparent window. A dysfunctional endothelium leads to corneal opacification, which is a common cause of corneal blindness worldwide and results in an unavoidable need for a transplantation. Unfortunately, the global donor shortage causes very long waiting lists. A pharmacological compound to stimulate in vivo corneal endothelial regeneration therefore is a very interesting alternative treatment to reduce the reliance on donor corneas. However, there are many limitations regarding the traditional drug discovery pipelines such as high cost and long development times. Moreover, the corneal anatomy hampers drug permeation. The aim of my PhD therefore is to tackle both these limitations so to deliver an innovative pharmacological treatment. Hence, this project proposes a high throughput biological screening of repurposed drugs, i.e. the screening of formerly approved compounds. This biological data will serve to develop and train a virtual compound screening model to predict additional potential lead compounds. The high throughput screenings together with a thorough characterization and optimalization of the physico-chemical properties of the main hits, will lead to the identification of one repurposed lead compound. Eventually, I will assemble this compound together with a corneal permeability enhancer into an inventive eye drop that facilitates corneal penetration to reach the endothelium.

Researcher(s)

• Promoter: Koppen Carina
• Co-promoter: Deben Christophe
• Co-promoter: Van den Bogerd Bert
• Fellow: Witters Charissa

Research team(s)

• Translational Neurosciences (TNW)

Project type(s)

• Research Project

Abstract

The corneal endothelium lies on the interior of the cornea, which is the window of our body. Proper function of this corneal endothelium is essential to obtain a crystal-clear cornea. Current consensus holds that endothelial cells do not display any significant regenerative capacity. Damaged or non-functional cells may consequently lead to corneal blindness. In this regard, stimulation of the regenerative capacity of the corneal endothelium is of exceptional importance in the search for new therapeutic possibilities. Furthermore, for in vitro biomedical research the culture of primary corneal endothelium is an extremely time-intensive process without full guarantee of cell expansion. In this project we intend to provide a solution for these issues by pharmacological stimulation of the regenerative capacity of corneal endothelial cells through re-orientation of available drugs. During this project various molecules will be subject to a tri-fold screening method (cellular, subcellular and molecular), which leads to the selection of a regenerative compound for corneal endothelial purposes. On the one hand, specific ROCK-inhibitors will be tested, given their growth potential within tissue regeneration. On the other hand, commercially available drug libraries will be screened for compounds with known activity for regenerative capacities.

Researcher(s)

• Promoter: Koppen Carina
• Co-promoter: Deben Christophe
• Co-promoter: Van den Bogerd Bert

Research team(s)

• Translational Neurosciences (TNW)

Project type(s)

• Research Project

Abstract

The cornea is the transparent window to the eye and as such, eye drops are an interesting route of drug administration. In this project, we propose the development of a 3D corneal cell model that can be used to investigate corneal drug interaction. Studies that aim to simulate the pharmacokinetics and toxicological properties of drugs are mainly based on oversimplified 2D monocultures or animal studies that suffer from interspecies differences. These limitations skew proper predictive power during preclinical drug investigation. We propose the development of a 3D corneal model that includes all three cell layers and integrated microfluidics to simulate relevant physiological flows such as the tear film. In this way, we reduce the need for animal studies, while simultaneously introducing the in vivo complexity of 3D cell environments. The materials used will be fabricated (photocrosslinkable biopolymers) and printed in-chip using 2PP bioprinting that can simultaneously integrate corneal cells. The interfaces between materials and corneal cells are characterized in depth using molecular spectroscopy techniques, while detailed protein expression of cells in the 3D ECM are benchmarked to ex vivo cadaveric donor corneas with proteomics. TEER, flow rate and cell viability will be measured real-time by integrated sensors. The project finally aims for a proof-of-concept to determine permeability coefficients of common ocular drugs in the fully assembled 3D cell culture chip.

Researcher(s)

• Promoter: Koppen Carina
• Co-promoter: Van den Bogerd Bert

Research team(s)

• Translational Neurosciences (TNW)

Project type(s)

• Research Project

Abstract

The cornea is a barrier that protects the eye from the outside world and likewise hampers drug absorption. Most ophthalmic medicine is washed away during instillation and is absorbed into systemic circulation. This is especially relevant when considering the main target population, the elderly, which also have an increased susceptibility to adverse drug reactions due to polypharmacy and decreased renal function. Hence, new topical formulations require rigorous testing to ensure therapeutic efficacy, while keeping local and systemic toxicity to a minimum. However, promising preclinical results are often not corroborated during human testing because available corneal models have poor predictive power. Traditional 2D culture fails to recapitulate complex tissues such as the cornea while animal models exhibit interspecies differences that limit translatability of results to humans. Organs-on-chips, which are rationally-designed microfluidic chips that contain artificial tissue, hold the promise of improving the status quo. While organ-on-chip technology has already proven its merits in certain fields, in the context of the cornea it remains relatively unexplored. This project proposal outlines the development of the first cornea-on-chip that comprises every cellular layer – epithelium, stroma and endothelium – of the cornea, exposed to the dynamics of an artificial tear film on one side and connected to an artificial anterior chamber on the other.

Researcher(s)

• Promoter: Koppen Carina
• Co-promoter: Van den Bogerd Bert
• Fellow: Van Meenen Joris

Research team(s)

• Translational Neurosciences (TNW)

Project type(s)

• Research Project

Abstract

The cornea is a multi-layered and transparent membrane that focusses incoming light on the lens. One of the layers is the endothelium, which is a monolayer of corneal endothelial cells (CEnCs) responsible for the clarity of the cornea. Several diseases can cause damage to this monolayer which might result in visual impairment or blindness. Corneal blindness is the 4th cause of blindness, affecting more than 10 million people worldwide. The main treatment comprises a corneal transplantation. However, there is a severe donor shortage since only 1 donor cornea is available for 70 people on the waiting list. The aim of this PhD proposal is to overcome the limitations regarding the low donor availability by developing a suitable donor tissue independent implant that can restore the endothelium. To this end, membranes will be produced on which lab-grown CEnCs will be seeded, after which the entire implant will be implanted into the cornea and restore its transparency. To develop a cell interactive and transparent scaffold that is strong enough for implantation, multiple approaches will be investigated and their performance will be compared. Single- and double-layered membranes that are cell interactive and mechanically strong enough will be produced. The required cell-interactivity will be provided by modified gelatines while poly(D,L-lactic acid) or poly (styrene-co-maleic anhydride) are chosen for their structural integrity.

Researcher(s)

• Promoter: Koppen Carina

Research team(s)

• Translational Neurosciences (TNW)

Project type(s)

• Research Project

Abstract

The eye is a biological tissue with optical and biomedical properties that govern the way the eye refracts light, focuses that light onto the retina and can dynamically alter that focus over a range of distances. This impressive flexibility results from the delicate way in which the mechanical properties of the eye very precisely affect its optics. These properties vary considerably between individuals and can alter over time in response to visual demands, as well as with eye growth, ageing and pathology. The origins of these biomechanical changes over time are very poorly understood, however, and point at a need for answers, given the increase in life expectancy and in societal demands for high quality vision. To address these issues, we present the first European collaboration that brings together a group of scientists that work on the optics and biomechanics of the eye, cover a broad range of disciplines and skills. This highly interdisciplinary consortium will also create a training network to give young researchers the opportunity to learn from renowned experts on ocular opto-mechanics, share their learning experiences and take advantage of placements in Universities, hospitals and industry. This will give them a wide and novel skill set to translate their research to scientific, industrial, or clinical applications, such as a new generation intraocular implants for cataract surgery, biologically relevant eye models that mimic the eye at any age, and novel treatment therapies that can control, reduce or ultimately prevent refractive error from occurring. These anticipated innovations will lead to wide-reaching and pioneering advances to enhance our understanding of the interrelationship between ocular optics and biomechanics. From this, the young researchers will emerge with multi-disciplinary, versatile skills, be highly employable, able to address skills shortages, be leaders in vision science and pioneer new industries in optical design and modelling.

Researcher(s)

• Promoter: Koppen Carina
• Co-promoter: Rozema Jos

Research team(s)

• Translational Neurosciences (TNW)

Project website

Project type(s)

• Research Project

Abstract

The rising prevalence of eye diseases and our increasing life expectancy has accelerated the demand for ophthalmic therapeutics. While there is a clear market for novel ophthalmic medicines, these drugs must also be safe. There are two standard approaches to preclinical drug testing; 1) the in vitro approach where human cells or cell lines are tested in cultures on mostly plasticware, and 2) in vivo testing using healthy animals or animal models of disease. Neither of these approaches are ideal as both deviate significantly from the true human in vivo status and the native environment of the cells. Moreover, side effects can present in human subjects that did not occur in either in vitro or in vivo models which can have devastating effects to the patients. Organ-on-chip (OoC) technology is an emerging biomedical field that aims to provide a 3-dimensional dynamic tissue environment to more closely model human physiology. OoC emerged from computer technology disciplines and aims to reproduce the smallest functional unit possible to represent the chemical, mechanical and functional aspects of human organs. These "smallest functional units" are microfluidic cell culture chips where multiple human cell types can grow together and interact as tissue would. The potential of OoC in Ophthalmology, and in particular the cornea, has not yet been properly exploited. The cornea is of distinct pharmacological interest, as the majority of ocular medications are administered as an eyedrop. Current ophthalmological in vitro models are limited to just one or two layers of the cornea. In reality, the cornea is a highly complex 'organ' consisting of five layers, each of which has a very specific physiology, functional role and cellular pathways. There is an unmet need to create a complete in vitro cornea model with adequate complexity. OoC technology allows to create a true cornea-on-a-chip including every corneal layer, mimicking the natural state as close as possible from the anterior chamber all the way through to the epithelium at an air-liquid interface, just like the surface of our eyes. The transparency of the cornea results from the complex interplay between the cells and the surrounding supporting structures. When one of the layers is compromised, it directly affects the integrity of the corneal system, possibly resulting in cellular damage or death which in turn directly impacts vision. Each layer of the cornea plays a role in the drug absorption process. The epithelial layer of the cornea is lipoidal in nature and acts as the first tissue barrier to drug absorption. On the other hand, the stroma is hydrophilic in nature and comprises 90% of the corneal thickness. Endothelium is the innermost layer separating barrier between the stroma and aqueous humour. This layer helps to maintain the corneal transparency due to its pump-and-leak mechanism that keeps the stroma in a relatively dehydrated state. In addition to difficulties related to the absorption of the drugs, metabolization through corneal enzymes also reduces drug bioavailability. The joined action of the three corneal layers maintains homeostasis and thus transparency. The drugs applied to treat eye-related conditions as well as those applied post-operatively (hypotensive drops, antibiotics, mydriatics and anti-inflammatory drugs) are applied topically and have to travel through the cornea where they could cause off-target damage. Therefore, the cornea model envisioned in this project includes the three main corneal layers: epithelium, stroma and endothelium. This is markedly different to existing in vitro models where mostly only the epithelium and in very few cases a part of the stroma is included. The aim of the project is to deliver a proof-of-concept for the design, production and validation of a cornea-on-chip model of the complete human cornea including all cell layers, that recreates the physiological environment precisely to adequately mimic the native human cornea.

Researcher(s)

• Promoter: Koppen Carina
• Fellow: Van Meenen Joris

Research team(s)

• Translational Neurosciences (TNW)

Project type(s)

• Research Project

Abstract

The corneal endothelium covers the inner surface of the cornea, the transparent window of the eye. When this cell layer gets damaged, this leads to painful blindness, necessitating transplantation. Currently, transplantation is limited by a severe donor shortage. That is why researchers aim to fabricate a lab grown tissue to treat blind patients and shorten waiting lists. However, my PhD project aims to develop an innovative membrane with the aim to eventually exploit in vivo regeneration without transplantation of cells. More specifically, this project includes the in vitro development of a membrane that is covered with miniscule patterns, which have 2 functions. On the one hand, the shape of the pattern itself will act as one-way signals that guide corneal endothelial cells to the middle of the cornea to restore its original barrier function. On the other hand, the patterns contain drugs that are specifically released when cells overgrow the patterns, thereby accelerating the process even further. I have 2 different strategies for its content, namely filling these patterns with either growth factors or exosomes secreted by stem cells. Eventually, I will establish a proof-of-principle in rabbits to prove the efficiency. Advantages are that this is a potential cost-effective off-the-shelf product that is safer for the patient as it does not involve any cells compared to cell therapy or transplantation and that the applicability stretches beyond the field of ophthalmology.

Researcher(s)

• Promoter: Koppen Carina
• Co-promoter: Ni Dhubhghaill Sorcha
• Fellow: Vercammen Hendrik

Research team(s)

• Translational Neurosciences (TNW)

Project type(s)

• Research Project

Abstract

The human eye is a unique, biological complex but vulnerable entity. It lacks protection of keratinized epithelium against infection and desiccation, as seen in almost every other area of the body. However, the ocular surface is specialised to protect the ocular structures and respond rapidly upon injury, while maintaining a smooth refractive surface to ensure visual acuity. One of the specialised cellular layers contributing to ocular tissue homeostasis is the conjunctiva. This thin mucous membrane belongs to the ocular surface epithelia, covering the sclera and the inside of the eyelids. In some ocular disorders, the conjunctiva is damaged, resulting in extensive scarring and inflammation, which can lead to several pathological conditions such as eyelid distortions, tear film disruptions, severe dry eyes, corneal ulcers and eventually blindness. The management of severe conjunctival surface disorders remains challenging for ophthalmologists worldwide. The conventional treatment comprises the surgical excision of the diseased conjunctiva. Normal wound healing post resection is based on epithelial migration from adjacent healthy conjunctiva to the wounded area. However, this healing process cannot take place in patients lacking sufficient healthy residual conjunctiva. Here, fibrosis and scar formation will occur, often reintroducing several pathological conditions as described above. Hence to avoid sequelae, the ocular surface requires reconstruction post excision using a cellularized conjunctival substitute. In this project, we aim to meet this unmet medical need by creating a cellularized conjunctival substitute for reconstructive surgery. By introducing fully synthetic self-assembling collagen-like-peptide hydrogels as carrier for human conjunctival-derived cells and eliminating all animal-derived components, we aim to provide a safe, consistent and functional conjunctival replacement. The graft's functionality will be tested in vitro by means of specifically designed tests for presence of conjunctival epithelial cells (barrier formation against infectious microbes), mucin-producing goblet cells (tear film stabilization) and stem cells (epithelium renewal).

Researcher(s)

• Promoter: Koppen Carina
• Fellow: Van Acker Sara

Research team(s)

• Translational Neurosciences (TNW)

Project type(s)

• Research Project

Abstract

Eye diseases are responsible for a huge economical burden globally, but are also associated with a drastic decrease in quality of life. Some of these diseases are associated to the loss of transparency of the window of the eye, namely the cornea. The cornea is the outermost part of the eye and is composed out of 3 different cell layers. The innermost layer, the corneal endothelium, maintains critical corneal hydration. Upon ageing, disease or trauma, this cell layer can be damaged to such an extent that the cornea swells and loses its transparency, which leads to blindness. Currently, the only treatment consists of full or partial transplantation of a donor cornea. Unfortunately, the supply does not meet the demand by far since only 1 donor is available per 70 patients. To overcome this limitation, the present project aims to develop a synthetic alternative that allows the efficient transplantation of healthy cells towards the site of tissue defect. To this end, biodegradable membranes will be developed using a combination of smart polyesters with shape memory effects, in combination with gelatin derivatives that mimic the cellular environment. These carriers will be seeded with cells, to allow transplantation to the site of tissue defect. Furthermore, the membranes will be analysed in depth both for mechanical properties as in vitro behavior prior to in vivo animal studies. Ideally, at the end of the project, the developed membrane should be ready for clinical trials.

Researcher(s)

• Promoter: Koppen Carina

Research team(s)

• Translational Neurosciences (TNW)

Project type(s)

• Research Project

Abstract

English Title: Tissue engineering for conjunctival reconstruction: Introducing self-assembled collagen-like-peptide scaffolds for the expansion of human conjunctival-derived cells in a xeno-free and serum-free environment. The human eye is a unique, biological complex but vulnerable entity. It lacks protection of keratinized epithelium against infection and desiccation, as seen in almost every other area of the body. However, the ocular surface is specialised to protect the ocular structures and respond rapidly upon injury, while maintaining a smooth refractive surface to ensure visual acuity. One of the specialised cellular layers contributing to ocular tissue homeostasis is the conjunctiva. This thin mucous membrane belongs to the ocular surface epithelia, covering the sclera and the inside of the eyelids. In some ocular disorders, the conjunctiva is damaged, resulting in extensive scarring and inflammation, which can lead to several pathological conditions such as eyelid distortions, tear film disruptions, severe dry eyes, corneal ulcers and eventually blindness. The management of severe conjunctival surface disorders remains challenging for ophthalmologists worldwide. The conventional treatment comprises the surgical excision of the diseased conjunctiva. Normal wound healing post resection is based on epithelial migration from adjacent healthy conjunctiva to the wounded area. However, this healing process cannot take place in patients lacking sufficient healthy residual conjunctiva. Here, fibrosis and scar formation will occur, often reintroducing several pathological conditions as described above. Hence to avoid sequelae, the ocular surface requires reconstruction post excision using a cellularized conjunctival substitute. In this project, we aim to meet this unmet medical need by creating a cellularized conjunctival substitute for reconstructive surgery. By introducing fully synthetic self-assembling collagen-like-peptide hydrogels as carrier for human conjunctival-derived cells and eliminating all animal-derived components, we aim to provide a safe, consistent and functional conjunctival replacement. The graft's functionality will be tested in vitro by means of specifically designed tests for presence of conjunctival epithelial cells (barrier formation against infectious microbes), mucin-producing goblet cells (tear film stabilization) and stem cells (epithelium renewal).

Researcher(s)

• Promoter: Koppen Carina
• Co-promoter: Tassignon Marie-Jose
• Fellow: Van Acker Sara

Research team(s)

• Translational Neurosciences (TNW)

Project type(s)

• Research Project

Abstract

Keratitis, or an inflammation of the cornea, is a common eye disease in which a biopsy of the cornea is required to determine its underlying cause which can range from infectious causes (including viral, bacterial, fungal, parasitic), mechanical (contact lens wear), non infectious (Vitamin A deficiency) (Gorski et al., 2015). Currently, no standardized tool is available for taking such biopsy and corneal scrapings are performed with a scalpel or wide bore needle, very often with inconclusive results as too little material is removed for fear of penetration. Consequently, biopsies are not performed as often and a broad-spectrum antibiotic is prescribed, assuming bacterial keratitis. The delay in providing optimal treatment can result in untoward sequelae such as corneal scarring and opacification requiring corneal transplantation. Collaborative research between the department of ophthalmology (UZA) Centre for Cell Therapy and Regenerative Medicine (Ophthalmology/Vaxinfectio – UA/UZA) and Product Development (UA) together with the industrial partner D.O.R.C. will lead to the development of a standardized tool for taking a biopsy of the cornea where the safety of the patient is guaranteed thereby addressing the aforementioned shortcomings.

Researcher(s)

• Promoter: De Bruyne Guido
• Co-promoter: Jacoby Alexis
• Co-promoter: Koppen Carina
• Co-promoter: Zakaria Nadia

Research team(s)

• Product development

Project type(s)

• Research Project

Abstract

Human corneal endothelial cells (HCEnC) regulate fluid and solute transport across the posterior surface of the human cornea and actively maintain the cornea in a dehydrated state, which is crucial for optical transparency.The dual function of the corneal endothelium is described as the "pump-leak hypothesis" which is essential to allow nutrition to the cornea whilst maintaining its avascularity and transparency. There is no evidence that human endothelial cells divide under normal circumstances as they are arrested in G1 phase of the cell cycle, although they can be induced to divide in vitro. When the amount of corneal endothelial cells decreases below a certain threshold, this cell layer can no longer pump sufficient fluid back to the anterior chamber, resulting in an irreversibly swollen, cloudy cornea. Despite its success, corneal transplantation (either full-thickness or partial) is limited worldwide by the shortage of suitable donor corneas incurring long waiting times. Initial progress to overcome this global shortage is the use of one donor cornea for multiple partial keratoplasties ("split-cornea transplantations"), by using one donor cornea for a partial endothelial and a stromal transplantation. This project aims to investigate ex vivo expansion of corneal endothelial cells to develop a cell sheet based therapy. This would overcome donor deficit that limits the treatment of corneal endotheliopathies. The principle is to expand primary human corneal endothelial cells isolated from human cadavers and to seed them on an ideal scaffolding material to introduce these cells in the patient. Specifically in this project we propose the expansion of human corneal endothelial cells (HCEnC) on human lens capsules to obtain a composite graft. The final goal of this project is a proof-of-principle of this functional cell sheet in a rabbit corneal endotheliopathy model.

Researcher(s)

• Promoter: Koppen Carina
• Promoter: Tassignon Marie-Jose
• Co-promoter: Zakaria Nadia
• Fellow: Van den Bogerd Bert

Research team(s)

• Translational Neurosciences (TNW)

Project type(s)

• Research Project

Abstract

During this PhD project, we want to gain insights in the pathophysiology of keratoconus (KC). Identification of the disease-causing genes is our primary goal. Afterwards we will investigate the role of these genes in the pathophysiology of KC and the biological processes in which they are involved.

Researcher(s)

• Promoter: Van Camp Guy
• Co-promoter: Koppen Carina
• Fellow: Valgaeren Hanne

Research team(s)

Project type(s)

• Research Project