Mesenchymal stem cells (MSC) have a well recognised potential for tissue repair. This potential is two pronged: they can differentiate into the functional cell types of the damaged tissues and they can support tissue recovery by secreting trophic factors, depositing an extracellular matrix (ECM) and dampening inflammation. Three-dimensional microscopy recently shown that MSCs in the bone marrow create an intricate proteo-cellular scaffold with the ECM forming an interconnected cellular continuum whose structure is guided by the deposited ECM. This proteo-cellular scaffold controls bone marrow functions from hematopoiesis to osteogenesis. In the current study we aimed to optimise ECM production under in vitro conditions by immortalised MSCs with the view that the generated ECM can be utilised for tissue repair. With immunocytochemistry we determined the deposition of bone marrow-characteristic ECM proteins: collagen I, III, IV, V, VI, laminin and fibronectin. While primary MSCs produced slightly higher amount ECM proteins than immortalised MSCs, the relative abundancy of the ECM proteins was very similar. In order to isolate the ECM, we optimised a decellularisation method based on gentle lysis with sodium-deoxycholate and DNase digestion. Immunostaining for collagen I, III, VI and fibronectin and labelling the nuclei with Hoechst-33342 confirmed removal of all cells while retaining the ECM in its original architecture. Ideally, the decellularised ECM retains associated cytokines and chemokines, such as CXCL12, important for attracting MSCs. To test this, we seeded Molm-13 leukemia cells on decellularised ECM as MSC-produced CXCL12- and other cytokines protect leukemia cells against chemotherapeutics. We found that the decellularisation process however removed these factors and thus for therapeutic purposes, the decellularised ECM would need to be re-loaded with the essential chemo/cytokines. Overall, we developed a system for decellularised ECM production by immortalised MSCs and the results warrant further exploration of this avenue.