Ideally, the adhered cellular aggregates will have a uniform size distribution and an array-formatted geometry. Further incubation allows the cellular aggregates to attach to the culture surface and start spreading and proliferating. During this process, PEG based hydrogel with the sloped walls enables detachment of cellular aggregate from the microwells and sedimentation onto the PS surface. After cellular aggregates have formed, the chip is inverted, which results in the cellular aggregates being plated onto a polystyrene (PS) culture surface in the same geometrical arrangement as the microwells. During this process, PEG based hydrogel with the sloped walls enhances the sedimentation of cells at the center of the microwells. The cells loaded into the chamber settle to the bottom of the microwells and form cellular aggregates. A microwell array with sloped walls made out of poly(ethylene glycol) (PEG) based hydrogel is placed in the bottom of the chamber, into which a single-cell suspension is loaded. Fig.1 1 illustrates the procedure for cultivating hiPS cells in the inverting microwell array chip. Here, we describe an inverting microwell array chip that is capable of producing uniform cellular aggregates and of controlling the position of the cellular aggregates in the adhesion culture. 29 However, the application was limited to single cell or small number of cells, and applicability to cellular aggregate is unclear. reported the BioFlipChip, a microfabricated micro well array chip, to trap small number of cells to pattern cells in a predetermined geometrical arrangement. 26, 27 However, the production of a geometrical arrangement of adhered cellular aggregates is still challenging previous reports adopted inoculation of cellular aggregates by pipetting, which resulted in adhesion at random positions.
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26, 27, 28 Couple of these methods realized retrieval of spheroids from the devices using medium flow. 21, 22, 23, 24, 25 In addition, some groups have reported microfluidic devices using gravity for spheroid formation. 15, 16, 17, 18, 19, 20, 21, 22, 23 Some groups have reported a subsequent adherent culture after formation of uniform cellular aggregates in microwells. In order to evaluate the effects of cellular aggregate size on differentiation, many previous studies have reported various methods for the formation of size-controlled cellular aggregates of hiPS and embryonic stem cells and the use of microwell structures, in particular, produced favorable results in the size-control. Although the break-down of cellular aggregates into single cells induces cell death, the formation of cellular aggregates promotes cell survival. Pipetting, which is generally the preferred method of handling liquids in biological laboratories, inevitably causes a broad distribution of cellular aggregate sizes, even in automated systems the size of cellular aggregates after pipetting depends on the shear stress, the size, and shape of the original aggregates, and the strength of the cell–cell and cell–extracellular matrix interactions of each individual aggregate.Ĭell-assembling of single hiPS cells offers a solution for producing uniform cellular aggregates. The conventional method for cultivating hiPS cells involves the handling of cells as cellular aggregates however, obtaining uniformly sized aggregates is technically difficult.
![sumaru illustartion sumaru illustartion](https://thumbs.dreamstime.com/b/summary-stamp-summary-round-grunge-stamp-summary-sign-summary-136961414.jpg)
7, 8 The development of processes, ideally involving programmed perfusion 9, 10, 11 and automated cultivation, 12, 13, 14 to control the culture microenvironment would overcome these problems and allow this approach to be applied to industrial applications. 6 In these fields, the maintenance of homogeneous cell populations and the reproducible control of differentiation are important issues, because behavior of hiPS cells is severely affected by the surrounding microenvironment, which includes the size and shape of the cellular aggregates during passage and differentiation, the geometrical arrangement of the adhered cellular aggregates (i.e., colonies), the timing of medium change as well as addition of differentiation factors, and composition of the extracellular matrix. The potency of Human induced pluripotent stem (hiPS) cells 1 promises huge changes in a variety of fields such as cell biology, 2 drug discovery, 3, 4, 5 and clinical therapy.