Development of a standardized manufacturing method of limbal stem cell grafts for curing corneal blindness
Abidine, Ousaamah (2023)
Abidine, Ousaamah
2023
Master's Programme in Biomedical Sciences and Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2023-10-23
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202310048640
https://urn.fi/URN:NBN:fi:tuni-202310048640
Tiivistelmä
This thesis work focuses on addressing the limitations of traditional treatment methods for limbal stem cell deficiency and introduces StemSight's advanced limbal stem cell graft. This research project encompasses several key aspects, including the refinement of the manufacturing platform's design. Quality assessment of the developed carrier was performed through a comprehensive analysis involving trypan blue staining, optical coherence tomography (OCT) imaging, rheological examination, and cell culture experiments, conducted both with and without aprotinin. Furthermore, immunocytochemistry was employed to assess marker proteins in the cultured cells.
The manufacturing protocol of the corneal graft carrier was developed in the course of this project with a clear vision of future automation possibilities. The manufacturing process involves a pressure-based method, allowing precise control over the shape and dimensions of the synthesized carrier. To examine the homogeneity of the fibrin solution, trypan blue was introduced. However, microscopic analysis revealed an uneven distribution of blue pigments, indicating a need for improved mixing techniques. OCT measurements established the mean thickness of the carrier at 198.4 ± 4.4 µm. In the context of limbal stem cell culture on the tissue-engineered corneal graft, results indicated greater ABCG2 expression in the absence of aprotinin, while Collagen IV expression was enhanced with the presence of aprotinin (5 µM). Rheological measurements of the carrier demonstrated stable storage and loss moduli G' and G'', consistent with hydrogel materials. However, the study identified challenges in the linear viscoelastic region, with an oscillation strain of approximately 1.4% and uneven strain at breakage, suggesting issues related to sample integrity and handling.
The outcomes achieved throughout the duration of this project have culminated in the establishment of an effective method for producing corneal graft carriers. Additionally, these results have provided valuable insights into potential areas of enhancement, including optimizing the mixing process for the components of the fibrin solution and exploring strategies to extend the product's shelf-life.
The manufacturing protocol of the corneal graft carrier was developed in the course of this project with a clear vision of future automation possibilities. The manufacturing process involves a pressure-based method, allowing precise control over the shape and dimensions of the synthesized carrier. To examine the homogeneity of the fibrin solution, trypan blue was introduced. However, microscopic analysis revealed an uneven distribution of blue pigments, indicating a need for improved mixing techniques. OCT measurements established the mean thickness of the carrier at 198.4 ± 4.4 µm. In the context of limbal stem cell culture on the tissue-engineered corneal graft, results indicated greater ABCG2 expression in the absence of aprotinin, while Collagen IV expression was enhanced with the presence of aprotinin (5 µM). Rheological measurements of the carrier demonstrated stable storage and loss moduli G' and G'', consistent with hydrogel materials. However, the study identified challenges in the linear viscoelastic region, with an oscillation strain of approximately 1.4% and uneven strain at breakage, suggesting issues related to sample integrity and handling.
The outcomes achieved throughout the duration of this project have culminated in the establishment of an effective method for producing corneal graft carriers. Additionally, these results have provided valuable insights into potential areas of enhancement, including optimizing the mixing process for the components of the fibrin solution and exploring strategies to extend the product's shelf-life.