conditions, complicated handling due to a large number of tubing connections, and the lack of compatibility with established laboratory infrastructure and bioassays. Performing experiments in desired cell culture microenvironments (2D or 3D model) possesses another additional challenge. The physiological relevance of a 3D microenvironment has been extensively studied elsewhere. [11,12] Recent studies show that, especially for clinically relevant processes, 2D cell culture systems have limitations as they result in abnormal phenotypes. [13] In addition to the biological issues, major technical hurdles exist with 2D cultivation in available microfluidic devices. These hurdles include immobilization of non-adherent cells such as cells derived from the hematopoietic system to prevent cell loss during medium exchange and detaching adherent cells from the chip for downstream analysis. Both procedures can substantially alter the inherent cell phenotype. [14,15] In comparison, 3D cell culture models gained significant relevance in the past years due to their biocompatibility, tissue like water content, high porosity, permeability, and in mimicking mechanical properties of the extracellular matrix resulting in a higher physiological relevance. [16] Despite its biological advantages, 3D cell culture hampers the combination of time-lapse data with endpoint measurements such as immunostainings as the hydrogel itself acts as a diffusion barrier. This diffusion barrier impedes supply with fresh nutrients, removal of waste products, and the implementation of efficient washing processes for endpoint staining protocols. In addition, embedding cells into macro 3D matrices complicates cell retrieval after cell cultivation as the hydrogel has to be removed enzymatically or chemically to release embedded cells. [17] In this work, we evaluate a new design of a macro-to-micro interface that can be used for simple and reliable control of microfluidic processes including comprehensive cell culture processes. The presented macro-to-micro interface overcomes significant technical challenges thereby making microfluidic cell culture procedures accessible for biological laboratories.By integrating a workflow that has been described previously, [6] we overcome mentioned limitations and are providing a valuable tool for cultivating single cells, cell-cell pairs, and low cell numbers in spherical hydrogel beads acting as a 3D microenvironment. At the same time, we combine time-lapse (fluorescence) microscopy data with endpoint measurements that provide A new design of a macro-to-micro interface that can be used for simple and reliable control of comprehensive microfluidic cell culture processes is introduced making microfluidic procedures easily accessible to biological laboratories. The novel macro-to-micro interface is evaluated by adapting a workflow for single-cell, cell pair, and cell cluster encapsulation into hydrogel beads acting as 3D microenvironment with subsequent long-term cultivation. For the first time, the coupling of single-cell...