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Cell therapies offer unquestionable promises for the treatment, and in some cases even the cure, of complex diseases. As we start to see more of these therapies gaining market authorization, attention is turning to the bioprocesses used for their manufacture, in particular the challenge of gaining higher levels of process control to help regulate cell behavior, manage process variability, and deliver product of a consistent quality. Many processes already incorporate the measurement of key markers such as nutrient consumption, metabolite production, and cell concentration, but these are often performed off-line and only at set time points in the process. Having the ability to monitor these markers in real-time using in-line sensors would offer significant advantages, allowing faster decision-making and a finer level of process control. In this study, we use Raman spectroscopy as an in-line optical sensor for bioprocess monitoring of an autologous T-cell immunotherapy model produced in a stirred tank bioreactor system. Using reference datasets generated on a standard bioanalyzer, we develop chemometric models from the Raman spectra for glucose, glutamine, lactate, and ammonia. These chemometric models can accurately monitor donor-specific increases in nutrient consumption and metabolite production as the primary T-cell transition from a recovery phase and begin proliferating. Using a univariate modeling approach, we then show how changes in peak intensity within the Raman spectra can be correlated with cell concentration and viability. These models, which act as surrogate markers, can be used to monitor cell behavior including cell proliferation rates, proliferative capacity, and transition of the cells to a quiescent phenotype. Finally, using the univariate models, we also demonstrate how Raman spectroscopy can be applied for real-time monitoring. The ability to measure these key parameters using an in-line Raman optical sensor makes it possible to have immediate feedback on process performance. This could help significantly improve cell therapy bioprocessing by allowing proactive decision-making based on real-time process data. Going forward, these types of in-line sensors also open up opportunities to improve bioprocesses further through concepts such as adaptive manufacturing.

Abstract A third of patients with critical limb ischemia (CLI) will eventually require limb amputation. Therapeutic neovascularization using unselected mononuclear cells to salvage ischemic limbs has produced modest results. The TIE2‐expressing monocytes/macrophages (TEMs) are a myeloid cell subset known to be highly angiogenic in tumours. This study aimed to examine the kinetics of TEMs in patients with CLI and whether these cells promote neovascularization of the ischemic limb. Here we show that there are 10‐fold more circulating TEMs in CLI patients, and removal of ischemia reduces their numbers to normal levels. TEM numbers in ischemic muscle are two‐fold greater than normoxic muscle from the same patient. TEMs from patients with CLI display greater proangiogenic activity than TIE2‐negative monocytes in vitro. Using a mouse model of hindlimb ischemia, lentiviral‐based Tie2 knockdown in TEMs impaired recovery from ischemia, whereas delivery of mouse macrophages overexpressing TIE2, or human TEMs isolated from CLI patients, rescued limb ischemia. These data suggest that enhancing TEM recruitment to the ischemic muscle may have the potential to improve limb neovascularization in CLI patients. →See accompanying articles http://dx.doi.org/10.1002/emmm.201302695 and http://dx.doi.org/10.1002/emmm.201302794

Abstract Anti‐angiogenic drugs are approved for the treatment of several cancer types, generally in the inoperable locally advanced or metastatic setting and in combination with other anti‐cancer agents. Recent clinical studies also suggest that anti‐angiogenic drugs can be useful in the pre‐operative (neoadjuvant) setting, by facilitating the shrinkage of the primary tumour and its surgical resection. However, the effects of neoadjuvant anti‐angiogenic therapy on the ability of tumours to form distant metastases are unclear. In this issue of EMBO Molecular Medicine, Ebos et al (2014) present carefully performed pre‐clinical studies in mice that analyse the effects of pre‐operative anti‐angiogenic therapy on tumour metastasis and survival.