Development of a three-dimensional in-vitro model of the human bone marrow using commercially available matrices and bone marrow derived mesenchymal stem cells/HS-5 stromal cell line

Kabrah, Saeed

Abstract

The function of the bone marrow is known to be affected by both haematopoietic malignancies as well as the chemotherapy used to treat them. To appropriately evaluate the role of disease versus treatment, this study aimed to develop a novel three-dimensional reproducible in-vitro culture system of the human bone marrow microenvironment using primary bone marrow mesenchymal stem cells (MSCs)/immortalised stromal cell line (HS-5) and commercially available novel matrices.
Initially, culture conditions and manipulation of the matrices required optimisation; cells growth and proliferation was evaluated in four different types of culture medium (low or high glucose-DMEM (DMEM-LG or -HG), DMEM/F12 and α-MEM), utilising four different concentrations of heat inactivated foetal bovine serum (0, 5, 11, and 17% for BM-MSCs, and 0, 5, 10 and 15% for HS-5), and also comparing supplementation with L-glutamine or Glutamax. Three different commercially available matrices (Nanofiber Biomerix and Alvetex scaffolds) have been employed in this study to evaluate the seeding and growth of the MSCs. A range of resuspension volumes (1 ml to 1 μl) was assessed over 30, 60, 90 and 120 minutes to ensure cells adhered to the scaffold and not the culture well. Seeding cells on the top of scaffolds were compared in stable and rotatory movement, also seeding using regular and vacuum centrifugations were tested. Due to the thickness and the density of the matrices, visualising MSCs is difficult, and several protocols including trypan blue and neutral red have been optimised to ensure clear visualisation of the cells. Retrieving MSCs from these scaffolds is essential to perform further analysis and evaluate cellular functionality; trypsin and Versene were compared to maximise the final yield with minimum cell damage. The final models for HS-5 were used to assess the cellular shape changes, proliferation, cell’s phenotype and quality, differentiation capacity, reference genes for expression studies and DNA stability. Moreover, the results of HS-5 functionalities were compared to the results of the traditional 2D culture flask. Also, cellular functionalities were compared in the presence and absence of the chemotherapeutic agent melphalan.
The medium evaluation showed that BM-MSCs grow better in DMEM-LG compared to DMEM-HG for HS-5, and the growth rate is directly proportional to increasing serum concentration, however, the optimum concentration was 11% and 10% for BM-MSCs and HS-5 respectively. Medium supplemented with L-glutamine showed better growth compared to those with Glutamax. The optimal conditions to set up the 3D cultures were determined. For the Biomerix scaffold, MSCs were loaded in 5 μl volumes and required 90 minutes to adhere while the Nanofiber matrices required 12.5 μl and 120 minutes, and Alvetex needed 50 μl and 90 minutes respectively. In term of seeding technique, results showed that dynamic seeding in rotatory movement induced spheroid formation and cellular clumping in high rotatory speed (60 - 100 rpm) while the low speed allowed cells to adhere to the well and form a monolayer. In addition, results indicated that static seeding is more suitable and did not reduce cellular viability. For cell retrieval from scaffolds, results indicated that trypsin is more effective than Versene. Evaluation of the HS-5 functionalities in these models showed that cells have a significant improvement in cell morphology, colony forming unit, differentiation and protein content compared to the traditional 2D cultures. Also, results indicated that 2D systems induced cell proliferation and reduced HS-5 quality. Results indicated that culture environment changed reference genes’ expressions and stabilities, where each 3D and 2D culture had different expression levels and stability values as compered by five different mathematical algorithms. Comparing melphalan treated and untreated cultures indicated that cells in 2D cultures were more susceptible to cellular damage and reduced cell quality and functionalities than in 3D culture.
In conclusion, these models enabled the investigation of MSCs proliferation, differentiation, gene expression, protein level and expression, and monitored changes in cellular behaviour between untreated and treated conditions. In addition, comparing these models with the traditional 2D culture technique confirmed the difference in cellular functionality between culture environments, including differences between different types of 3D models. This study highlights the importance of assessing the suitability of 3D models in studying MSCs in-vitro and mimicking the in-vivo situation by comparison with bone marrow from leukaemia patients in future research.

Citation

Kabrah, S. Development of a three-dimensional in-vitro model of the human bone marrow using commercially available matrices and bone marrow derived mesenchymal stem cells/HS-5 stromal cell line. (Thesis). University of the West of England. Retrieved from https://uwe-repository.worktribe.com/output/10596716

• Thesis Type: Thesis
• Deposit Date: Mar 30, 2023
• Publicly Available Date: Mar 30, 2023
• Public URL: https://uwe-repository.worktribe.com/output/10596716
• Award Date: Dec 31, 2016