1) Growth of more than one cell type, separated by an inter-space full of media.
2) Growth of several million cells, letting repeated cell sampling operations without interruption of the culture.
3) Separate sampling of the different cell types included in the co-culture.
4) Repeated media renewal and sampling operations without the interruption of the culture.
The biological product of this concept is the “Cystic 3D cell culture” that offers, in a single volume, at least three separate sheets of cells, with or without matrixes, growing with well defined borders, surrounding apocket (cyst) with 15 to 25 mL of shared media (see upper figure). The system is a cube like structure with 6 sides or 3 pairs of opposed walls facing each other. In this system the central cavity or cyst receives everything secreted or released by the surrounding cells including metabolites, extracellular organelles, exosomes, shedding microvesicles, apoptotic blebs and others. Cell shits are attached to the walls, uncoated or coated with specific matrix of single molecules such as Laminin, Vitronectin, Collagen, etc. or molecular mixtures such as Matrigel or others, all favoring cell attachment through integrins. One side of the device may be coated with a thick layer of semisolid gel allowing the formation of cell clusters and cell penetration evaluation (invasion assays).
Soluble secretions and particulate elements released by cells forming the walls circulate randomly through the cyst media, like in the blastocysts, ovary follicles and others. Those suspended elements become in contact with the other wall cells dragged by soft and slow convection streams, generated by controlled temperature gradients,. Many physiologic reactions are controlled by soluble molecules and particulate elements, such as exosomes released from living cells
With an accurate control of hypoxia, close to physiologic DO (about 2 ppm), CO2 control and dehydration control (Petaka G3 conditions), these cultures could evolve for more than 2 weeks, allowing long term inter-cellular interactions. The cyst culture system (Petaka G3 device) allows for free-enzyme cell release of all the cells or samples of each cell type separately, therefore sampling each side independently for microscopy, FACS, PCR or biochemistry analysis.
The bottom side layer of cells is preferably influenced by particulate elements, it receives by gravity sedimentation all those elements having density over 1, released by the upper cells; this influence is even more intensive on all cells living on the side of the cyst when exposed to a multiplied g force vector, if the device is subject to a convenient centrifugation.