The use of engineered cells, tissues, and organs has the opportunity to change the way injuries and diseases are treated. Commercialization of these groundbreaking technologies has been limited in part by the complex and costly nature of their manufacture. Process-related variability and even small changes in the manufacturing process of a living product will impact its quality. Without real-time integrated detection, the magnitude and mechanism of that impact are largely unknown. Real-time and non-destructive sensor technologies are key for in-process insight and ensuring a consistent product throughout commercial scale-up and/or scale-out. The application of a measurement technology into a manufacturing process requires cell and tissue developers to understand the best way to apply a sensor to their process, and for sensor manufacturers to understand the design requirements and end-user needs. Furthermore, sensors to monitor component cells’ health and phenotype need to be compatible with novel integrated and automated manufacturing equipment. This review summarizes commercially relevant sensor technologies that can detect meaningful quality attributes during the manufacturing of regenerative medicine products, the gaps within each technology, and sensor considerations for manufacturing.

During limb development a cluster of mesodermal cells in the distal posterior limb bud, the zone of polarizing activity (ZPA), regulates patterning along the anterior‐posterior axis. The signaling molecule responsible for this patterning is Sonic hedgehog (Shh), which is controlled by a highly conserved limb‐specific enhancer, the ZPA regulatory sequence (ZRS).ZRS microdeletion results in limbs that fail to express Shh and do not develop posterior limb elements, whereas many ZRS single nucleotide variants (SNVs) lead to anterior ectopic Shh expression causing preaxial polydactyly (PPD). However, the ZRS mechanism of action is not well characterized. The ZRS contains putative binding sites for Hand2, Twist1, and Hoxd13: transcription factors (TFs) known to regulate Shh. Both Hand2 and Twist1 are basic helix‐loop‐helix (bHLH) TFs and are thought to function as homo‐ or heterodimers, while Hoxd13 has been shown to interact with Hand2.The present study's objective is to determine the role of these TFs in ZRS function. We hypothesize that altering Hand2, Twist1, and Hoxd13 bindings sites, either alone or in concert, will disrupt ZRS activity. To evaluate enhancer activity, ZRS was cloned into an enhancer‐GFP reporter construct. Hand2, Twist1, and Hoxd13 binding sites were altered using site‐directed mutagenesis, and constructs were electroporated into the presumptive limb buds of chicken embryos (Hamburger‐Hamilton stage 14). GFP expression was evaluated 48 hours post‐electroporation. We determined that mutating the binding sites for all three TFs in concert resulted in loss of ZRS activity. However, constructs with either Twist1 or Hoxd13 binding sites intact remained active. These data suggest that Twist1 and Hoxd13 may have either redundant or temporally distinct roles in ZRS regulation of Shh. Future work will determine whether these TFs bind the ZRS at the specific sites we have identified.

The ZPA regulatory sequence (ZRS) is a limb‐specific enhancer that localizes Sonic Hedgehog (SHH) expression to the zone of polarizing activity (ZPA), which consequently regulates anteroposterior patterning in the developing limb. ZRS deletion lacks SHH expression in the limb resulting in loss of posterior limb structures, while ZRS single nucleotide variants (SNVs) can lead to ectopic SHH expression causing preaxial polydactyly (PPD). However, the ZRS mechanism of action is not well characterized. HAND2 and HOXD13 are key transcription factors in SHH regulation that bind to the ZRS, potentially modulating SHH transcription. The present study’s objective was to determine the role of HAND2 and HOXD13 binding sites in ZRS function. To evaluate ZRS enhancer activity, ZRS was cloned into an enhancer‐GFP reporter construct. HAND2 and HOXD13 binding sites were altered using site‐directed mutagenesis, and contructs were electroporated into presumptive limb buds in Hamburger‐Hamilton stage 14 chicken embryos. GFP expression was evaluated 48 hours post‐electroporation. ZRS with mutated HAND2 and HOXD13 binding sites retained activity in the limb bud in a pattern that overlapped ZRS activity. This is unexpected since HAND2 and HOXD13 are known to be critical for SHH expression. Future work is needed to clarify the binding site(s) critical to activity and to characterize the factors involved in ZRS function. Support or Funding Information Supported in part by a grant from the Department of Pathology, Loma Linda University.