Recreating the microenvironment of the neurovascular unit

• The neurovascular unit (NVU) comprises the blood-brain-barrier (BBB) and its surrounding astrocytes, pericytes and neurons that are embedded in the extracellular matrix (ECM). As the main function of the BBB is to protect the brain from inlet of pathogens and toxins, the specialized endothelial cells that keep the barrier tight will also hinder the passage of pharmaceuticals. Understanding the detailed microenvironment and cellular interactions involved in the development of the neurovascular unit is, therefore, an important step towards designing CNS-targeting pharmaceuticals that can pass into the brain. At the same time, the initial steps of pharmaceutical development often involve the use of animal based in vitro models with poor human translation; thus, there is a great need for novel methods to better mimic the complexity of the human NVU. Apart from conventional cell culture models, the use of micro-engineered devices, microphysiological systems (MPS), have gained popularity. The use of MPS allows for fabrication of tissue-like structures using stem cells and provide more in vivo-like parameters in terms of physical cues and dynamic flow. Various materials have been explored for chip fabrication, and biological and synthetic ECM-mimicking hydrogels have been developed for cell encapsulation. Unfortunately, models developed to date often lack either: i) relevant and reproducible cell sources, ii) materials that allow for easy chip fabrication where sensors can be integrated to understand metabolic effects and barrier integrity, or iii) animal-free defined ECM-mimicking scaffolds that support the culture of sensitive cells. This thesis presents an isogenic model of the BBB using iPSC-derived endothelial cells and astrocytes cultured in a MPS made from the non-absorbing polymer OSTE+ that allows for easy fabrication and integration of interdigitated gold electrodes for continuous barrier integrity monitoring. The model presents barrier-protective effects of the BBB-penetrating drug NACA. To better understand the metabolic attributes of astrocytes, a flow-cell sensor is evaluated for the measurement of glucose and lactate turnover during a ketogenic diet. The results imply that such a sensor is valuable for the measurement of metabolic changes and can, in the future, be integrated into MPSs.Furthermore, a model of early neuronal development is realized by using defined copper-free click chemistry to conjugate laminin to a hyaluronic-based hydrogel system for the differentiation of neuroepithelial stem cells. The use of the hydrogel is validated for bioprinting, and the first-ever printed neuroepithelial stem cells are presented. In another study astrocyte 3D culture and bioprinting is evaluated in peptide conjugated hyaluronic-based hydrogels. Unique attachment and spreading of human fetal astrocytes is observed while the common glioblastoma U87 cells display a rounded up morphology. The results of the hydrogel study imply that the defined chemistry of the hydrogel is suitable for both neuroepithelial stem cells, U87 and fetal primary astrocytes, and can in the future be integrated into MPS to circumvent the use of animal derived matrices. In summary, these results provide solutions to some of the problems to date and lay the ground work for the continuation of the development of human-relevant MPS of the NVU.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Neurovetenskaper (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Neurosciences (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinsk bioteknologi -- Biomaterialvetenskap (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Medical Biotechnology -- Biomaterials Science (hsv//eng)

Nyckelord

microphysiological systems

neurovascular unit

induced pluripotent stem cells

extracellular matrix

hydrogels

in vitro models

mikrofysiologiska system

neurovaskulära enheten

inducerade pluripotenta stamceller

extracellulära matrisen

hydrogeler

in vitro modeller

Bioteknologi

Biotechnology

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