CUSTOM 3D ORGANOIDS

CMA TECHNOLOGY APPLIED ON 3D CELL CULTURE SYSTEMS

 

Cell MicroArray (CMA) platform is a high-throughput experimental method that can be exploited to study 3-D cell culture systems. Cells cultured as 3-D models exhibit features that are closer to the in vivo conditions (Vinci et al., 2012) and have proven to recapitulate the in vivo conditions. This characteristic is mainly due to the behaviour of cells when grown in matrices and scaffolds. In the area of stem cell research, 3-D cultures have widely been studied particularly to engineer tissue constructs in addition to basic research (good source for the continuous supply of specifically differentiated cells) and therapeutic applications  (tissue engineering) and / or drug testing (study the synergistic effects of biologically important substances on cells).

The Application of the CMA technology in stem cell research, and in particular in 3-D cultures during the differentiation of induced Pluripotent Stem Cells, is an alternative approach. The semi-automatic process (CMA platform), is a high-throughput analysis tool, to rapidly and simultaneously characterize the hiPSC, could provide an opportunity to minimizing time-saving and reagent cost-efficient. Currently, hiPSCs can be routinely expanded as cell suspension in 3-D culture conditions. Traditional approches to screen their pluripotent and differentiation potential are cumbersome. Thus, the CMA technology could steamline the whole process by analyzing hundreds samples at the same time (La Spada A et al., 2014).

Cell suspension is fixed in formalin and embedded in paraffin. The paraffin blocks are then used as donor blocks in the different CMA constructions and the slides obtained from the CMA are immunostained and the bona fide clone is then used for the down the line need.

Artificial tissue block (CMA) was constructed using several hiPS cells that were cultured as 3-D cell aggregates simultaneously the CMA contains different hIPS clones, and culture conditions representative of the pluripotency and differentiative status. CMA blocks were sliced using a microtome and the slides were H&E and immunofluorescence (IF) stained. In details this methodological approch combining CMA & 3-D cultures preserves cell morphology and allows to better characterize protein expression and visualize the sub-cellular localization of the markers more comprehensively. The figure below shows the Tuj1 (anti-beta-III-tubulin immunofluorescence, red) and Hoechst 33258 (blue) counterstaining of neural rosettes derived from human pluripotent stem cell differentiation grown in three dymensional (3D) conditions. 3D aggregates were processed following the Cell MicroArray technology (CMA) using the Galileo CK4500 (Integrated Systems Engineering) platform.


Galileo platform coupled to immunocytochemistry techniques, allows the study of 3D cellular aggregates and organoids. The image below shows the complexity and heterogeneity in the composition of each spheroid analyzed.