Raman spectroscopic analysis of cell differentiation and death modes

Raman spectroscopy provides opportunities for non-invasive, non-destructive, label-free analysis of cell states based on changes in the biochemical composition of cells. We are investigating the suitability of Raman spectroscopy to assess the stages of human embryonic stem cell (hESC) differentiation towards pancreatic insulin-positive cells. Raman microspectrometry analysis has revealed macromolecular composition differences over time that distinguished cell populations differentiating to pancreatic cell types, such as by an increase in the protein-to-nucleic acid signal ratio and to distinguish the presence of insulin. Added insight into these macromolecular changes were provided by principal component analysis (PCA) of the data. However, the application of PCA can be difficult to interpret. The usefulness of non-negative matrix factorization was explored to improve the interpretability of overlapping Raman bands. We demonstrated the utility of this procedure by analyzing spectra to determine the cellular insulin or glucagon content. Thus, Raman spectroscopy can detect such differences in cells to detect the desired product as well as the potential to detect residual hESCs or the emergence of unwanted cells. We also investigated the suitability of Raman spectroscopy to detect the onset and types of cell death. Apoptotic, necrotic or autophagic Chinese Hamster Ovary cells were compared to uninduced cultures using Raman spectroscopy and PCA. Furthermore, uninduced cells were compared to cells sorted at different stages of apoptosis to determine how early the onset of apoptosis could be detected. Changes were observed in several peaks during the course of cell death, with repeated changes observed in nucleic acid- and lipid-associated peaks, enabling the distinction of cell death modes. Application of such death monitoring capabilities to cellular therapy cultures should be even more useful, given the need for more process analytical technologies to address the often more variable performance of these cultures, especially when adaptive control is needed for primary cell derived manufacturing

Process analytical utility of Raman spectroscopy for cell therapy manufacturing

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Acoustic cell washing and raman spectroscopy technologies To address cell therapy bioprocess challenges

Many organizations are confronting the challenges of economically ensuring the manufacture of safe and efficacious cell therapy products. These processes often depend on devices and methods that were developed for only related applications, such as blood cell processing or scientific research. Thus, we are in a window of opportunity to tailor innovative technologies to address the emerging specialized needs of cell therapy manufacturing. The most frequent unit operation is to wash cells between process stages, such as from DMSO containing cryopreservation medium to culture expansion medium. In particular for relatively small-scale autologous cell therapy processing, cell washing is imperfectly performed by closed system blood cell centrifuges or filters. We previously developed an acoustic cell separation device, widely used for over 15 years in CHO cell perfusion cultures. This technology acts as a non-fouling filter for months of operation, by using the forces generated in ultrasonic standing wave fields. These forces separate cells from medium based on differences in density and compressibility. Greater than 99.9% cell washing with 95% washed cell recovery efficiencies have been provided by our device. We also have recently enhanced the acoustic technology to perfuse 100 million cell/mL cultures, maintaining \u3e99% cell separation efficiencies. This provides an alternative high performance closed manufacturing system, to perfuse, concentrate and wash cells, with no physical filter barrier or mechanical moving parts. While many clinical trials have had few adverse events, the great promise of cellular therapies comes with grave risks, such as from potentially oncogenic pluripotent cells present in embryonic stem cell derived populations. There is an urgent need for process analytical technologies to non-invasively monitor mammalian cell populations and improve the reliability of manufactured cell products. This includes to monitor both the expected differentiation as well as to detect unexpected cells in the process. Recently, technological advances have led to an explosive growth in the capabilities of Raman spectroscopy, increasing the potential for novel applications. We are developing the use of this spectroscopic technique to track cell development, by measuring macromolecular changes in cell samples from cultures where stem cells are differentiated towards insulin-producing cells for the treatment of diabetes. Raman spectroscopy has great potential to provide continuous on-line assessment of cell quality during the manufacture of cell-derived therapeutic cells

Real time and delayed effects of subcortical low intensity focused ultrasound.

Deep brain nuclei are integral components of large-scale circuits mediating important cognitive and sensorimotor functions. However, because they fall outside the domain of conventional non-invasive neuromodulatory techniques, their study has been primarily based on neuropsychological models, limiting the ability to fully characterize their role and to develop interventions in cases where they are damaged. To address this gap, we used the emerging technology of non-invasive low-intensity focused ultrasound (LIFU) to directly modulate left lateralized basal ganglia structures in healthy volunteers. During sonication, we observed local and distal decreases in blood oxygenation level dependent (BOLD) signal in the targeted left globus pallidus (GP) and in large-scale cortical networks. We also observed a generalized decrease in relative perfusion throughout the cerebrum following sonication. These results show, for the first time using functional MRI data, the ability to modulate deep-brain nuclei using LIFU while measuring its local and global consequences, opening the door for future applications of subcortical LIFU

Neuroanatomical differences in the memory systems of intellectual giftedness and typical development.

IntroductionStudying neuro-structural markers of intellectual giftedness (IG) will inform scientific understanding of the processes helping children excel academically.MethodsStructural and diffusion-weighted MRI was used to compare regional brain shape and connectivity of 12 children with average to high average IQ and 18 IG children, defined as having IQ greater than 145.ResultsIG had larger subcortical structures and more robust white matter microstructural organization between those structures in regions associated with explicit memory. TD had more connected, larger subcortical structures in regions associated with implicit memory.ConclusionsIt was found that the memory systems within brains of children with exceptional intellectual abilities are differently sized and connected compared to the brains of typically developing children. These different neurodevelopmental trajectories suggest different learning strategies. A spectrum of intelligence types is envisioned, facilitated by different ratios of implicit and explicit system, which was validated using a large external dataset

A community-partnered approach for diversity in COVID-19 vaccine clinical trials.

IntroductionCommunities of color have faced disproportionate morbidity and mortality from COVID-19, coupled with historical underrepresentation in US clinical trials, creating challenges for equitable participation in developing and testing a safe and effective COVID-19 vaccine.MethodsTo increase diversity, including racial and ethnic representation, in local Los Angeles County NIH-sponsored Phase 3 SARS-CoV-2 vaccine clinical trials, we used deliberative community engagement approaches to form a Community Consultant Panel (CCP) that partnered with trial research teams. Thirteen members were recruited, including expertise from essential workers, community-based and faith-based organizations, or leaders from racial and ethnic minority communities.ResultsWorking closely with local investigators for the vaccine studies, the CCP provided critical insight on best practices for community trust building, clinical trial participation, and reliable information dissemination regarding COVID-19 vaccines. Modifying recruitment, outreach, and trial protocols led to majority-minority participants (55%-78%) in each of the three vaccine clinical trials. CCP's input led to cultural tailoring of recruitment materials, changes in recruitment messaging, and supportive services to improve trial accessibility and acceptability (transportation, protocols for cultural competency, and support linkages to care in case of an adverse event). Barriers to clinical trial participation unable to be resolved included childcare, requests for after-hours appointment availability, and mobile locations for trial visits.ConclusionUsing deliberative community engagement can provide critical and timely insight into the community-centered barriers to COVID-19 vaccine trial participation, including addressing social determinants of health, trust, clinical trial literacy, structural barriers, and identifying trusted messenger and reliable sources of information