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Establishing an optimized experimental framework to model the multiple myeloma microenvironment for targeted drug screening
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Resumo: O mieloma múltiplo (MM) é uma neoplasia das células plasmáticas (PC), resultante da expansão clonal destas células na medula óssea (BM). O estabelecimento de sistemas ex vivo que reflitam de forma fidedigna este microambiente é crucial para o desenvolvimento de novas terapias. Este trabalho teve como objetivo criar uma estrutura experimental otimizada, capaz de manter células derivadas da BM de doentes em cultura de curta duração, permitindo o estudo dos efeitos de fármacos no microambiente do MM.
A investigação teve início com experiências de co-culturas utilizando a linha celular MM1R e células mononucleares do sangue periférico (PBMCs) de doadores saudáveis, avaliadas em condições 2D e 3D Matrigel® por 72h. As PBMCs demonstraram boa viabilidade geral, enquanto as células MM1R apresentaram declínio consistente na viabilidade após 24h em cultura. BM estabeleceu que
200.000 eventos eram necessários para preservar as proporções populacionais, garantindo, ao mesmo tempo, pelo menos 20 eventos por subconjunto. Com base nesse padrão, três modelos foram comparados: modelo 2D, modelo Matrigel 3D e o modelo 3DTEBM, sob diferentes condições. Entre estes, o 3DTEBM a uma concentração de 1 x 106 células/mL proporcionou a maior viabilidade geral, mantendo também a capacidade proliferativa por até 48h.
Por fim, um painel otimizado de citometria de fluxo foi cuidadosamente desenvolvido, incluindo titulações de anticorpos para garantir a identificação fiável de populações imunes importantes. Em conjunto, este projeto estabelece um fluxo de trabalho sólido baseado em 3DTEBM para cultura de curto prazo de amostras de pacientes com MM, criando uma base para futuras aplicações de testes de fármacos num microambiente clinicamente relevante.
Abstract: Multiple Myeloma (MM) is a plasma cell (PC) malignancy resulting from the clonal expansion of PCs in the bone marrow (BM). Establishing ex vivo systems that closely reflect this microenvironment is crucial for therapeutic development. This work aimed to create an optimized framework capable of sustaining patient-derived BM cells in short-term culture to enable the study of drug effects within the MM microenvironment. The investigation began with co-culture experiments using the MM1R cell line and peripheral blood mononuclear cells (PBMCs) from healthy donors, assessed under both two-dimensional (2D) and three-dimensional (3D) Matrigel® conditions for 72h. PBMCs demonstrated overall good viability, whereas MM1R cells consistently showed a decline in viability after 24h in culture. To determine optimal sampling requirements, an in silico downsampling analysis of five BM samples established that 200,000 events were necessary to preserve population proportions while ensuring at least 20 events per subset. Based on this standard, three models were compared: 2D culture, 3D Matrigel, and the 3D Tissue- Engineered Bone Marrow (3DTEBM) system, under different conditions. Among these, the 3DTEBM seeded at 1 x 106 cells/mL provided the highest overall viability while also maintaining proliferative capacity for up to 48h. Finally, an optimized flow cytometry panel was carefully developed, including antibody titrations to ensure reliable identification of key immune populations. Collectively, this work establishes a strong 3DTEBM based workflow for short term culture of MM patient samples, creating a foundation for future drug-testing applications in a clinically relevant microenvironment.
Abstract: Multiple Myeloma (MM) is a plasma cell (PC) malignancy resulting from the clonal expansion of PCs in the bone marrow (BM). Establishing ex vivo systems that closely reflect this microenvironment is crucial for therapeutic development. This work aimed to create an optimized framework capable of sustaining patient-derived BM cells in short-term culture to enable the study of drug effects within the MM microenvironment. The investigation began with co-culture experiments using the MM1R cell line and peripheral blood mononuclear cells (PBMCs) from healthy donors, assessed under both two-dimensional (2D) and three-dimensional (3D) Matrigel® conditions for 72h. PBMCs demonstrated overall good viability, whereas MM1R cells consistently showed a decline in viability after 24h in culture. To determine optimal sampling requirements, an in silico downsampling analysis of five BM samples established that 200,000 events were necessary to preserve population proportions while ensuring at least 20 events per subset. Based on this standard, three models were compared: 2D culture, 3D Matrigel, and the 3D Tissue- Engineered Bone Marrow (3DTEBM) system, under different conditions. Among these, the 3DTEBM seeded at 1 x 106 cells/mL provided the highest overall viability while also maintaining proliferative capacity for up to 48h. Finally, an optimized flow cytometry panel was carefully developed, including antibody titrations to ensure reliable identification of key immune populations. Collectively, this work establishes a strong 3DTEBM based workflow for short term culture of MM patient samples, creating a foundation for future drug-testing applications in a clinically relevant microenvironment.
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Multiple myeloma Bone narrow Bone marrow microenvironment Cell culture Flow cytometry Drug tests