Development of cell culture models for multiple myeloma drug discovery: comparison of traditional 2D, Transwell 2D, and 3D-bioprinted settings
Location
Suwanee, GA
Start Date
7-5-2024 1:00 PM
End Date
7-5-2024 4:00 PM
Description
Introduction: Multiple myeloma (MM) is a plasma cell disorder accounting for about 10% of hematological malignancies worldwide. Despite advances in treatment, including hematopoietic stem cell transplants and high-dose chemotherapy, the median survival remains at roughly three years post-diagnosis and long-term remissions are rare. One of the main reasons that this disease is hard to cure is due to the protection of bone marrow microenvironment. Therefore, developing a realistic in vitro model especially by applying three dimensional (3D) bio-printed setting is more valuable than a traditional 2D model in drug discovery screening. Bortezomib, a dipeptide boronic acid, serves as chemotherapeutic inhibitor of the 20S and 26S proteasome and used as part of the initial therapies for transplant-eligible patients of MM. Apoptotic activation is a well-supported hypothesis in myeloma cells treated with bortezomib. This study aims to evaluate the time and dosage-dependent inhibitory effects of bortezomib on MM cell growth in a traditional 2D, a Transwell 2D, and a bio-printed 3D cell culture model.
Methods: MM cells were treated with bortezomib (0 – 25 nM) for 24, 48, or 72 hours, and Presto Blue assays were applied to measure cell viability in traditional 2D cell culture model. A 2D Transwell model was established by adding human bone marrow stromal HS-5 cells in the basal chambers and MM RPMI8226 cells in the apical chamber. The effects of bortezomib (0 – 40 nM) on apoptosis induction in MM RPMI8226 cells were evaluated for the protein expression of apoptotic markers (caspase-3 and PARP-1) through western blotting. 3D model constructs were made by mixing cells and cell viable alginate based bio-inks and using the Celllink 3D bioprinter. Constructs are comprised of a co-culture of RPMI8226 and HS-5 cells to simulate a rustic bone marrow microenvironment. Constructs were treated with Live/Dead permeable dyes and imaged after drug treatment to assess viability.
Results: Our data showed that bortezomib decreased cell viability in both dose- and time-dependent manners. The IC50 values were determined to be 5.2, 2.1, and 1.3 nM after 24, 48, and 72 hr treatment of bortezomib, respectively. Additional results within the transwell models have exhibited a dose-dependent increase in the expression of cleaved caspase-3 and PARP-1 that peaks at 20 nM but to a lesser extent compared to the 2D model. The 3D constructs exhibited the similar level of decreased RPMI8226 cell viability with a degree of difference when compared to HS-5 cells.
Conclusions: We used bortezomib as a model drug to evaluate its inhibitory effect on the growth of MM cells in three different cell culture settings. With the change from 2D to 3D models, the apoptogenic effects of bortezomib were reduced but still to a significant level compared to the control.
Embargo Period
5-23-2024
Development of cell culture models for multiple myeloma drug discovery: comparison of traditional 2D, Transwell 2D, and 3D-bioprinted settings
Suwanee, GA
Introduction: Multiple myeloma (MM) is a plasma cell disorder accounting for about 10% of hematological malignancies worldwide. Despite advances in treatment, including hematopoietic stem cell transplants and high-dose chemotherapy, the median survival remains at roughly three years post-diagnosis and long-term remissions are rare. One of the main reasons that this disease is hard to cure is due to the protection of bone marrow microenvironment. Therefore, developing a realistic in vitro model especially by applying three dimensional (3D) bio-printed setting is more valuable than a traditional 2D model in drug discovery screening. Bortezomib, a dipeptide boronic acid, serves as chemotherapeutic inhibitor of the 20S and 26S proteasome and used as part of the initial therapies for transplant-eligible patients of MM. Apoptotic activation is a well-supported hypothesis in myeloma cells treated with bortezomib. This study aims to evaluate the time and dosage-dependent inhibitory effects of bortezomib on MM cell growth in a traditional 2D, a Transwell 2D, and a bio-printed 3D cell culture model.
Methods: MM cells were treated with bortezomib (0 – 25 nM) for 24, 48, or 72 hours, and Presto Blue assays were applied to measure cell viability in traditional 2D cell culture model. A 2D Transwell model was established by adding human bone marrow stromal HS-5 cells in the basal chambers and MM RPMI8226 cells in the apical chamber. The effects of bortezomib (0 – 40 nM) on apoptosis induction in MM RPMI8226 cells were evaluated for the protein expression of apoptotic markers (caspase-3 and PARP-1) through western blotting. 3D model constructs were made by mixing cells and cell viable alginate based bio-inks and using the Celllink 3D bioprinter. Constructs are comprised of a co-culture of RPMI8226 and HS-5 cells to simulate a rustic bone marrow microenvironment. Constructs were treated with Live/Dead permeable dyes and imaged after drug treatment to assess viability.
Results: Our data showed that bortezomib decreased cell viability in both dose- and time-dependent manners. The IC50 values were determined to be 5.2, 2.1, and 1.3 nM after 24, 48, and 72 hr treatment of bortezomib, respectively. Additional results within the transwell models have exhibited a dose-dependent increase in the expression of cleaved caspase-3 and PARP-1 that peaks at 20 nM but to a lesser extent compared to the 2D model. The 3D constructs exhibited the similar level of decreased RPMI8226 cell viability with a degree of difference when compared to HS-5 cells.
Conclusions: We used bortezomib as a model drug to evaluate its inhibitory effect on the growth of MM cells in three different cell culture settings. With the change from 2D to 3D models, the apoptogenic effects of bortezomib were reduced but still to a significant level compared to the control.