PB2099 PATIENT‐SPECIFIC MULTIPLE MYELOMA MODEL USING 3D PRINTED ORGANOIDS FOR PREDICTING AND UNDERSTANDING TREATMENT RESPONSE
Authors: C. Rodriguez Valdes, J. Aleman Hernandez, M. A. Nikiforov, G. Almeida‐Porada, C. Porada, S. George, A. Maycock, A. Skardal
Journal: HemaSphere (2019)

Background: Results of studies using current pre‐clinical multiple myeloma (MM) models are limited in their ability to incorporate the effects bone marrow microenvironment has on cell proliferation, cell death, and drug resistance. 3D constructs, or organoids, have recently been tested to better recapitulate MM tumors and their microenvironment using a combination of commercially available and primary cell cultures. Here we present the first reported viable patient‐derived organoids composed entirely from fresh tumor cells co‐cultured with the patient's own stromal stem cells. This patient‐specific model serves as a platform to study tumor behavior and treatment screening. Aims: We aim to construct a durable 3D in vitro model derived exclusively from patient's own myeloma and stromal cells to serve as a true patient‐specific model. We examined for adequate representation of the bone marrow microenvironment throughout the life of the organoid and feasibility of drug screening. Methods Primary cells Bone marrow (BM) aspirates were collected from MM patients undergoing treatment. In the first 24 hours, samples were treated with Ficoll‐paque solution for cell sorting. CD138+ cells were positively selected and isolated using coated magnetic beads. The CD138+ cells were then fluorescently tagged with nanocrystals into the cytoplasm. Total BM pellet and assorted cell count and viability was quantified by the NucleoCounter NC‐200 automated cell counter. 3D Organoids The isolated stromal and multiple myeloma cells were incorporated into an ECM‐based hydrogel system and high throughput bioprinted into 3D patient‐specific disk‐shaped organoids. The bioengineered set up consisted of a 96 well plate, were 10 μl of the ECM/cell mixture was dispensed. Each organoid consisted of 200,000 cells (cell density of 20 × 10 6 cell/ml). Each well was then exposed to UV for hydrogel crosslinking and organoids covered in tissue culture media that was changed on day 2 and 4. Drug treatment studies Organoids were screened in parallel with established treatment regimens (Figure 1). Their viability was assessed by live/dead and ATP metabolism assays, and corroborated by H&amp;E staining. The results described were performed in triplicate. Immunohistochemistry Parafin blocks of organoids were obtained at each evaluation time point using CD138+ for plasma cells, CD11b+ for stromal cells, and additional CD45+ for lymphocyte infiltrates. Results: We were able to build a microenvironment to sustain patient stromal cells and fresh tumor cells that allowed 3D models to live long enough for tumor studies and chemotherapy screening. A minimum of 6cc of aspirate is sufficient to bioprint a 96 well plate experiment per patient. Average time from sample aspiration to organoid development to initiation of drug protocols was 24 hours, with a metabolic viability of 7 days. Each group of patient‐specific organoids exhibited a mixed response to the three stablished therapies, but a low variability inside each regiment. Summary/Conclusion: Our organoid constructs provide the first reported in vitro patient‐specific 3D MM model that mimics in vivo environmental and cellular composition of a patient tumor niche. By maintaining patient‐derived organoids viable for 5‐7 days, chemotherapy screening to guide patient care could be a possibility in the near future. The ability to sort cells prior to performing organoid constructs had no impact on organoid viability and could allow for studies such as gene editing, CAR‐T therapy, or niche studies combining different cell types. Further testing is needed to optimize cell viability of different cell types. image