Primary Chronic Lymphocytic Leukemia Cells Can be Maintained Long-Term in Serum-Free, Cytokine-Free 3D Culture

Chronic Lymphocytic Leukemia (CLL) cells undergo rapid and spontaneous apoptosis when cultured in vitro, challenging the study of disease biology. Conventional CLL culture platforms are dependent on addition of high concentrations of soluble molecules, animal- derived serum, adhesion proteins or allogeneic/xenogeneic stromal cells. We have hypothesized that a three-dimensional (3D) architecture may create a niche-like culture system, enabling CLL cell maintenance within a self-perpetuating microenvironment, in the absence of additional growth factors.

In order to assess optimal culture parameters, several conditions were tested: (1) Initial seeding densities (2, 10 and 40 x 10⁶ cells/scaffold); (2) Culture media (IMDM or RPMI + 10% FBS, StemSpan SFEM and Hybridoma SFM); (3) Oxygen tension (21% or 5% O₂) and (4) Scaffold coating (uncoated, collagen, RGD). Primary mononuclear cells were seeded onto polyurethane scaffolds and complete media exchanges were performed every third day. The system has enabled successful culture for up to 54 days in 6 out of 7 primary patient samples, 5 of them isolated from peripheral blood (PB) and 1 from bone marrow (BM). Conventional cultures, set up in parallel, died out by day 7.

On days 1, 7, 14 and 28, cultures were evaluated for viable cell expansion in the scaffold with Cell Titer Glo 3D (CTG), for morphological characteristics of CLL lymphocytes by May-Grunwald Giemsa (MGG) staining and for in situ niche-like structure formation with scanning electron microscopy (SEM) and fluorescence confocal microscopy. In both serum-containing media (RPMI and IMDM), cultures established with 2 x 10⁶ cells/scaffold showed a reduction in CTG at day 28 compared with that at day 1 (p<0.01, N=2, n=2), whereas higher seeding densities maintained ATP content and metabolism in situ, suggesting improved maintenance of viable cells in scaffolds. For higher seeding densities, SEM images of scaffolds at day 28 show cells clustering in niches and MGG-stained cells show morphological characteristics of CLL, similar to those isolated from patients at day 0. Cultures with 1 x 10⁷ cells/scaffold in hypoxia (5% O₂) and in StemSpan SFEM also showed reduction in CTG from days 1 to 28 (p<0.01, N=3, n=2), suggesting lack of in situ proliferation in these conditions. Furthermore, functionalization of scaffolds with collagen or RGD coating did not improve culture dynamics, displaying no difference between conditions at days 1 or 28 with CTG and created more debris and cellular anomalies when visualised with MGG. In contrast, CLL cells morphologically identical to those isolated from the patient were retrieved from the uncoated scaffolds.

When comparing RPMI + 10% FBS (FBS) and serum-free media Hybridoma SFM (SF), primary CLL cells were seeded at 1 x 10⁷ cells/scaffold. Scaffold-extracted and supernatant cells were counted and analysed for viability with Guava Via Count assay and assessed for morphology with MGG at every medium exchange. At days 1, 14 and 28 of culture, cells extracted from scaffolds were also analysed with propidium iodide and Annexin V staining for detection of early apoptosis. In the first 8 days of culture, 10.2% of seeded cells egressed from FBS culture, compared with 5.9% in SF, both stabilizing thereafter with similar numbers of viable cells migrating into the supernatant. After 8 days, cells were more viable in SF than FBS in the supernatant (43.3% and 21.8%, respectively; p=0.0097) as well as when cells extracted from scaffolds on days 14 (61.9% and 32.6%, respectively; p<0.0001) and 28 (61% and 16%, respectively; p=0.0013). Consistent with these results, in situ proliferation was higher for SF than FBS (n=3, p<0.05). Using confocal microscopy, DAPI, and an amine reactive dye, widespread in situ scaffolddistribution of cells was observed in SF by day 28 with formation of cellular clusters inside the scaffold. Furthermore, lymphocytes and nurse-like cells (vimentin positive) were detected at day 28 in SF by confocal microscopy in niche-like structures.

To the best of our knowledge, this work constitutes the first long-term culture (up to 8 weeks) of patient-derived primary CLL cells without cytokines, serum or feeder layers by using a 3D scaffold and high cell density. This platform may address an unmet need for a culture system which could represent the native disease environment, and a potential drug-testing platform.