We previously demonstrated that “stemness” of human hematopoietic progenitor cells (HPC) was maintained in a co-culture setting with a monolayer of human mesenchymal stromal cells (MSC). To simulate and monitor the marrow microenvironment of the HPC niche more precisely we have established a 3D co-culture system based on a proprietary KITChip.

The KITChip was developed by the Karlsruhe Institute of Technology (KIT) and represents a unique microchip with defined microwell cavities for 3D cell cultures. Sample acquisition was approved by the local Ethics Committee and informed written consent was obtained from all subjects. MSC were derived from human bone marrow of healthy voluntary donors, and HPC were isolated from umbilical cord blood. Cells were mixed in suspension in a ratio of 3:2 (3x105 MSC and 2x105 HPC) and inoculated into the KITChip, which was subsequently mounted into a microbioreactor. This closed loop setup allowed precise control of medium flow and oxygen saturation. After 1 to 5 days of co-culture, the two cell populations were analyzed by immunostaining, RT2-PCR and colony formation assay.

MSC form a complex 3D mesh in the microcavities of the KITChip and were maintained stable for up to 6 weeks. We have demonstrated that HPC were distributed three-dimensionally inside this MSC mesh and could be kept viable in this environment for at least 14 days. A defined proportion of CD34+ HPC adhered to the MSC in the microcavities and built up direct cellular connections to the surrounding MSC. By means of RT2-PCR, we could demonstrate that throughout the whole culture period of 14 days a subpopulation of CD34+/p21+/CXCR4+ cells was maintained in the 3D-environment more efficiently than compared to conventional co-culture with MSC monolayer. This was confirmed by Western blotting after the isolation of both cell populations from the chip. The colony formation assay revealed that the plasticity of the HPC cultivated in the 3D KITChip was nearly the same as that of freshly isolated HPC at day 0, whereas HPC co-cultured on MSC monolayer showed a significant decrease in stem cell plasticity. Further analysis under hypoxic conditions (5% O2) indicated that gene expressions of CD33, CD34, CD38 and CD44 were markedly reduced, while those of CD90, CD105, c-Kit, p21, SDF-1 and Angpt-1 remained stable compared to normoxic culture conditions.

This novel model system allows analysis of the major determinants of the niche and the impact of a 3D microenvironment on vital stem cell functions. Early HPC were maintained more efficiently and showed a superior plasticity potential when cultured in the 3D KITChip as compared to conventional 2D co-culture systems. Current studies are in process to define the functional significance of the observed changes in gene expression pattern under hypoxic conditions, which resembles the physiologic milieu of the marrow.


Wuchter:ETICHO: Consultancy, Honoraria; Sanofi: Honoraria for lectures Other. Ho:Sanofi-Genzyme: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Author notes


Asterisk with author names denotes non-ASH members.