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Towards 3D bio-printed spinal cord organoids
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics and Neurobiology.ORCID iD: 0000-0002-9558-5501
2024 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

The development of 3D bioprinting technology has provided a new direction for the replacement of organs or tissues and the development of drug testing models. Testing cell adhesion, proliferation, and differentiation in different printed scaffolds for creating functional 3D bio-printed structures provides the possibility of establishing a patient-specific in vitro model for neurodegenerative diseases. This thesis aims to establish a 3D bio-printed spinal cord model for drug research of ALS by exploring the factors affecting cell adhesion, growth, and differentiation in different hydrogels, and the suitable printing conditions.

In Paper I, we compared the adhesion and cell survival rates of BCs on the surfaces of the scaffolds with different stiffness and different chemical covering substracts and found the effects of physical and chemical factors for cell adhesion, proliferation, and differentiation through comparison, which can be used as a reference for exploring the conditions for further 3D printing mixing with cells inside. 

In Paper II, gelatin-based hydrogel was selected as the main material for printing the scaffold. By testing the survival rate of BCs in the different concentrations of gelatin with different concentrations of crosslinker, we selected a protocol that is suitable for cell viability, cell differentiation, and bioprintability. Unfortunately, when this protocol is applied to hiPSCs, it can obtain the viability of cells after printing, but cell differentiation was only observed on the surface of the scaffolds since cells in the middle of the printed structure lack contact with the surrounding culture medium.

Paper III showed that BCs attracted endothelial cells sprouting from aortic rings in their co-cultured 3D-printed scaffolds and guided the migration direction of endothelial cells. Also, after implantation at the injury DRTZ, they helped with vascularization by increasing the blood vessel volume and vessel diameters.

In Paper IV, we improved the protocol from Paper II for hiPSCs-derived MNs by reducing the concentration of gelatin and adding MSP loaded with cintrofin and gliafin. Two printable methods that could keep the printed structures during culturing were tested, and one was chosen for further printing based on cell viability during bio-ink preparation. A lower concentration of gelatin helped with getting better access to the surrounding culture medium and achieving motor neuron differentiation inside the scaffolds.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2024. , p. 42
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 2097
Keywords [en]
3D bioscaffold, gelatin, iPSC, differentiation
National Category
Neurosciences
Research subject
Biology
Identifiers
URN: urn:nbn:se:uu:diva-540590ISBN: 978-91-513-2278-0 (print)OAI: oai:DiVA.org:uu-540590DiVA, id: diva2:1906519
Public defence
2024-12-05, A1:107, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2024-11-12 Created: 2024-10-17 Last updated: 2024-11-12
List of papers
1. Effect of scaffold properties on adhesion and maintenance of boundary cap neural crest stem cells in vitro
Open this publication in new window or tab >>Effect of scaffold properties on adhesion and maintenance of boundary cap neural crest stem cells in vitro
2020 (English)In: Journal of Biomedical Materials Research. Part A, ISSN 1549-3296, E-ISSN 1552-4965, Vol. 108, no 6, p. 1274-1280Article in journal (Refereed) Published
Abstract [en]

Optimal combination of stem cells and biocompatible support material is a promising strategy for successful tissue engineering. The required differentiation of stem cells is crucial for functionality of engineered tissues and can be regulated by chemical and physical cues. Here we examined how boundary cap neural crest stem cells (bNCSCs) are affected when cultured in the same medium, but on collagen- or laminin-polyacrylamide (PAA) scaffolds of different stiffness (0.5, 1, or similar to 7 kPa). bNCSCs displayed marked differences in their ability to attach, maintain a large cell population and differentiate, depending on scaffold stiffness. These findings show that the design of physical cues is an important parameter to achieve optimal stem cell properties for tissue repair and engineering.

Place, publisher, year, edition, pages
Wiley, 2020
Keywords
differentiation, neural crest stem cell, polyacrylamide, scaffold, survival
National Category
Cell and Molecular Biology Neurosciences
Identifiers
urn:nbn:se:uu:diva-424393 (URN)10.1002/jbm.a.36900 (DOI)000514635600001 ()32061005 (PubMedID)
Funder
Swedish Institute
Available from: 2020-11-11 Created: 2020-11-11 Last updated: 2024-10-17Bibliographically approved
2. Towards 3D Bioprinted Spinal Cord Organoids
Open this publication in new window or tab >>Towards 3D Bioprinted Spinal Cord Organoids
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2022 (English)In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 23, no 10Article in journal (Refereed) Published
Abstract [en]

Three-dimensional (3D) cultures, so-called organoids, have emerged as an attractive tool for disease modeling and therapeutic innovations. Here, we aim to determine if boundary cap neural crest stem cells (BC) can survive and differentiate in gelatin-based 3D bioprinted bioink scaffolds in order to establish an enabling technology for the fabrication of spinal cord organoids on a chip. BC previously demonstrated the ability to support survival and differentiation of co-implanted or co-cultured cells and supported motor neuron survival in excitotoxically challenged spinal cord slice cultures. We tested different combinations of bioink and cross-linked material, analyzed the survival of BC on the surface and inside the scaffolds, and then tested if human iPSC-derived neural cells (motor neuron precursors and astrocytes) can be printed with the same protocol, which was developed for BC. We showed that this protocol is applicable for human cells. Neural differentiation was more prominent in the peripheral compared to central parts of the printed construct, presumably because of easier access to differentiation-promoting factors in the medium. These findings show that the gelatin-based and enzymatically cross-linked hydrogel is a suitable bioink for building a multicellular, bioprinted spinal cord organoid, but that further measures are still required to achieve uniform neural differentiation.

Place, publisher, year, edition, pages
MDPIMDPI AG, 2022
National Category
Nano Technology
Research subject
Engineering Science with specialization in Nanotechnology and Functional Materials
Identifiers
urn:nbn:se:uu:diva-475031 (URN)10.3390/ijms23105788 (DOI)000804307600001 ()35628601 (PubMedID)
Available from: 2022-05-29 Created: 2022-05-29 Last updated: 2024-12-03Bibliographically approved
3. Boundary cap neural crest stem cells promote angiogenesis after transplantation to avulsed dorsal roots in mice and induce migration of endothelial cells in 3D printed scaffolds
Open this publication in new window or tab >>Boundary cap neural crest stem cells promote angiogenesis after transplantation to avulsed dorsal roots in mice and induce migration of endothelial cells in 3D printed scaffolds
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2024 (English)In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 826, article id 137724Article in journal (Refereed) Published
Abstract [en]

Dorsal root avulsion injuries lead to loss of sensation and to reorganization of blood vessels (BVs) in the injured area. The inability of injured sensory axons to re-enter the spinal cord results in permanent loss of sensation, and often also leads to the development of neuropathic pain. Approaches that restore connection between peripheral sensory axons and their CNS targets are thus urgently need. Previous research has shown that sensory axons from peripherally grafted human sensory neurons are able to enter the spinal cord by growing along BVs which penetrate the CNS from the spinal cord surface. In this study we analysed the distribution of BVs after avulsion injury and how their pattern is affected by implantation at the injury site of boundary cap neural crest stem cells (bNCSCs), a transient cluster of cells, which are located at the boundary between the spinal cord and peripheral nervous system and assist the growth of sensory axons from periphery into the spinal cord during development. The superficial dorsal spinal cord vasculature was examined using intravital microscopy and intravascular BV labelling. bNCSC transplantation increased vascular volume in a non-dose responsive manner, whereas dorsal root avulsion alone did not decrease the vascular volume. To determine whether bNCSC are endowed with angiogenic properties we prepared 3D printed scaffolds, containing bNCSCs together with rings prepared from mouse aorta. We show that bNCSC do induce migration and assembly of endothelial cells in this system. These findings suggest that bNCSC transplant can promote vascularization in vivo and contribute to BV formation in 3D printed scaffolds.

Place, publisher, year, edition, pages
Elsevier, 2024
Keywords
Dorsal root, Spinal cord injury, Angiogenesis, Neural stem cell, Transplantation, 3D printing
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-528694 (URN)10.1016/j.neulet.2024.137724 (DOI)001219155300001 ()38467271 (PubMedID)
Funder
Swedish Research Council, 2018-02314Swedish National Space Board, 2021–0005Swedish Society for Medical Research (SSMF)Göran Gustafsson Foundation for promotion of scientific research at Uppala University and Royal Institute of Technology
Available from: 2024-05-31 Created: 2024-05-31 Last updated: 2024-10-17Bibliographically approved
4. Differentiation of human motor neurons in a 3D-printed scaffold - a step towards standardized and personalized human spinal cord tissue for modeling motor neuron diseases
Open this publication in new window or tab >>Differentiation of human motor neurons in a 3D-printed scaffold - a step towards standardized and personalized human spinal cord tissue for modeling motor neuron diseases
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(English)Manuscript (preprint) (Other academic)
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-540589 (URN)
Available from: 2024-10-17 Created: 2024-10-17 Last updated: 2024-10-17

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