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Identification of Key Chemical and Physical Factors for Development of Better Cell Models of the Human Brain

Boross, Andreas LU (2021) KBKM05 20201
Pure and Applied Biochemistry
Theoretical Chemistry
Abstract
Simulating the natural environment of living tissue with in vitro cell culture models has paved the way for scientific discoveries, and provided valuable insight into areas such as drug discovery and regenerative medicine. A new emerging technique of culturing cells on biomimetic three-dimensional scaffolds stands as a promising alternative to the traditional way of culturing cells on two-dimensional surfaces. Consisting of a network of sub-micrometer sized fibers, these scaffolds are able to capture the complexities of living tissue in a better fashion, and thereby offer more realistic models of the human body. In addition to studying the effects of substrate topography on cellular development, we were interested in how cell... (More)
Simulating the natural environment of living tissue with in vitro cell culture models has paved the way for scientific discoveries, and provided valuable insight into areas such as drug discovery and regenerative medicine. A new emerging technique of culturing cells on biomimetic three-dimensional scaffolds stands as a promising alternative to the traditional way of culturing cells on two-dimensional surfaces. Consisting of a network of sub-micrometer sized fibers, these scaffolds are able to capture the complexities of living tissue in a better fashion, and thereby offer more realistic models of the human body. In addition to studying the effects of substrate topography on cellular development, we were interested in how cell differentiation was affected by the presence of the extracellular matrix protein laminin, and how the actions of myosin II regulate cell fate and morphology. With the aim of identifying key physical and chemical cues in cellular development to produce more physiologically relevant assays, we here cultured pluripotent human neural progenitor cells on fiber scaffolds produced through electrospinning, and exposed cultured cells to laminins and the myosin II-inhibiting drug blebbistatin. We found that, although there were small variations in cell differentiation for different laminin isoforms, laminins did not have a significant effect on the potential of progenitor cells to differentiate into neurons or glial cells. While inhibiting myosin II also did not have a significant effect on cell differentiation, it did promote the growth of neurites and helped preserve a rounder morphology of cultured cells, demonstrating that blebbistatin may be useful to maintain stable neuronal-like cell cultures. These results further establish fibrous scaffolds as a suitable model to investigate cellular behaviour, and provide deeper understanding of relevant cues for the design of tailor-made cell cultures. (Less)
Popular Abstract
Scientists often create laboratory models that simulate various processes of the human body. The aim of these models may be to gain better understanding of a disease, develop therapeutic treatments, or evaluate the effects of drugs in development. In order to achieve accurate test results, it is important to design these models such that they resemble the natural environment as closely as possible. However, this is not as easy as it may seem. Living tissue consists of a complex three-dimensional structure of macromolecules, with a wide variety of proteins that help cells grow and carry out their tasks.

With the aim of capturing the topographical environment in the body, a new cell culturing technique has been developed that utilizes an... (More)
Scientists often create laboratory models that simulate various processes of the human body. The aim of these models may be to gain better understanding of a disease, develop therapeutic treatments, or evaluate the effects of drugs in development. In order to achieve accurate test results, it is important to design these models such that they resemble the natural environment as closely as possible. However, this is not as easy as it may seem. Living tissue consists of a complex three-dimensional structure of macromolecules, with a wide variety of proteins that help cells grow and carry out their tasks.

With the aim of capturing the topographical environment in the body, a new cell culturing technique has been developed that utilizes an interconnected network of thin fibers as a substrate for cells to grow on. These fiber networks, known as nanofiber scaffolds, are considered a preferable alternative to traditional cell culturing, which is usually performed on two-dimensional surfaces. The nanofiber scaffold allows cells to grow and migrate in all three dimensions, and thus represents a more accurate resemblance to the natural environment. Nanofibers are typically produced through electrospinning, wherein an applied voltage is used to extract thin threads of polymer solution from a syringe, which are dried as they travel through the air, and are deposited on a collector plate.

Along with matching the physical structure of living tissue, it is desired to match the biochemical environment. An essential type of proteins present in the extracellular space are laminins, which contribute to the overall structure of tissue, and are involved in the growth and differentiation of cells. Laminins are assemblies of chains of proteins, and exist as multiple genetic variants, each of which may have slightly different functions. Human-derived laminins have not been available for research use up until fairly recently. It is thus of great interest to investigate how the development and differentiation of cells may be affected when grown in the presence of laminin.

An important tool in cell culture studies on the nervous system are progenitor cells. Similarly to stem cells, progenitor cells are pluripotent, meaning that they can differentiate into multiple different cell types. However, progenitor cells are slightly more restricted in what types of cells they can differentiate into, and have a limited capacity for self-renewal.

In this master thesis, we studied how laminins and nanofiber scaffolds affect the viability, differentiation, and morphology of progenitor cells. Additionally, we investigated how cells are affected by myosin, a family of proteins responsible for cellular contraction. Cells originating from the forebrain of a human embryo were grown on laminin-coated nanofibers, and in the presence of blebbistatin, a drug that inhibits the activity of myosin.

Our results show that progenitor cells survive well and can differentiate into both neurons and supporting glial cells under the present conditions. Although we did not observe a significant difference in differentiation potential by either laminin or nanofiber scaffolds, small fluctuations in differentiation capacity indicate that they may still have an effect. More encouragingly, cells treated with the myosin inhibitor blebbistatin showed a distinct change in morphology, with a rounder cell shape and more outgrowths. It appears that relieving cells of tension has a favourable effect on their growth; indicating that blebbistatin is a useful tool for maintaining natural morphology in cultured cells for longer periods of time. (Less)
Please use this url to cite or link to this publication:
author
Boross, Andreas LU
supervisor
organization
course
KBKM05 20201
year
type
H2 - Master's Degree (Two Years)
subject
keywords
brain model, cell culture, nanofibers, electrospinning, neural progenitor cells, differentiation, cell morphology, laminin, blebbistatin, myosin, immunocytochemistry, applied biochemistry
language
English
id
9044418
date added to LUP
2021-05-20 14:02:13
date last changed
2021-05-20 14:02:13
@misc{9044418,
  abstract     = {{Simulating the natural environment of living tissue with in vitro cell culture models has paved the way for scientific discoveries, and provided valuable insight into areas such as drug discovery and regenerative medicine. A new emerging technique of culturing cells on biomimetic three-dimensional scaffolds stands as a promising alternative to the traditional way of culturing cells on two-dimensional surfaces. Consisting of a network of sub-micrometer sized fibers, these scaffolds are able to capture the complexities of living tissue in a better fashion, and thereby offer more realistic models of the human body. In addition to studying the effects of substrate topography on cellular development, we were interested in how cell differentiation was affected by the presence of the extracellular matrix protein laminin, and how the actions of myosin II regulate cell fate and morphology. With the aim of identifying key physical and chemical cues in cellular development to produce more physiologically relevant assays, we here cultured pluripotent human neural progenitor cells on fiber scaffolds produced through electrospinning, and exposed cultured cells to laminins and the myosin II-inhibiting drug blebbistatin. We found that, although there were small variations in cell differentiation for different laminin isoforms, laminins did not have a significant effect on the potential of progenitor cells to differentiate into neurons or glial cells. While inhibiting myosin II also did not have a significant effect on cell differentiation, it did promote the growth of neurites and helped preserve a rounder morphology of cultured cells, demonstrating that blebbistatin may be useful to maintain stable neuronal-like cell cultures. These results further establish fibrous scaffolds as a suitable model to investigate cellular behaviour, and provide deeper understanding of relevant cues for the design of tailor-made cell cultures.}},
  author       = {{Boross, Andreas}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Identification of Key Chemical and Physical Factors for Development of Better Cell Models of the Human Brain}},
  year         = {{2021}},
}