Optogenetic methods for pacing of cardiac tissue can be realized by direct genetic modification of the cardiomyocytes to express light-sensitive actuators, such as channelrhodopsin-2, ChR2, or by introduction of light-sensitized non-myocytes that couple to the cardiac cells and yield responsiveness to optical pacing. In this study, we engineer three-dimensional “spark cells” spheroids, composed of ChR2-expressing human embryonic kidney cells (from 100 to 100,000 cells per spheroid), and characterize their morphology as function of cell density and time. These “spark-cell” spheroids are then deployed to demonstrate site-specific optical pacing of human stem-cell-derived cardiomyocytes (hiPSC-CMs) in 96-well format using non-localized light application and all-optical electrophysiology with voltage and calcium small-molecule dyes or genetically encoded sensors. We show that the spheroids can be handled using liquid pipetting and can confer optical responsiveness of cardiac tissue earlier than direct viral or liposomal genetic modification of the cardiomyocytes, with 24% providing reliable stimulation of the iPSC-CMs within 6 h and >80% within 24 h. Moreover, our data show that the spheroids can be frozen in liquid nitrogen for long-term storage and transportation, after which they can be deployed as a reagent on site for optical cardiac pacing. In all cases, optical stimulation was achieved at relatively low light levels (<0.15 mW/mm 2 ) when 5 ms or longer pulses were used. Our results demonstrate a scalable, cost-effective method with a cryopreservable reagent to achieve contactless optical stimulation of cardiac cell constructs without genetically modifying the myocytes, that can be integrated in a robotics-amenable workflow for high-throughput drug testing.
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Simultaneous Ca 2+ Imaging and Optogenetic Stimulation of Cortical Astrocytes in Adult Murine Brain Slices
Astrocytes are actively involved in a neuroprotective role in the brain, which includes scavenging reactive oxygen species to minimize tissue damage. They also modulate neuroinflammation and reactive gliosis prevalent in several brain disorders like epilepsy, Alzheimer's, and Parkinson's disease. In animal models, targeted manipulation of astrocytic function via modulation of their calcium (Ca2+) oscillations by incorporating light‐sensitive cation channels like Channelrhodopsin‐2 (ChR2) offers a promising avenue in influencing the long‐term progression of these disorders. However, using adult animals for Ca2+imaging poses major challenges, including accelerated deterioration of
- NSF-PAR ID:
- 10238445
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols in Neuroscience
- Volume:
- 94
- Issue:
- 1
- ISSN:
- 1934-8584
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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This article was corrected on 25 July 2022. See the end of the full text for details.
Basic Protocol 1 : Genetic engineering strategy for the generation of modular light‐activated protein dimerization unitsSupport Protocol 1 : Molecular cloningBasic Protocol 2 : Cell culture and transfectionSupport Protocol 2 : Production of dark containers for optogenetic samplesBasic Protocol 3 : Confocal microscopy and light‐dependent activation of the dimerization systemAlternate Protocol 1 : Protein recruitment to intracellular compartmentsAlternate Protocol 2 : Induction of organelles’ membrane tetheringAlternate Protocol 3 : Optogenetic reconstitution of protein functionBasic Protocol 4 : Image analysisSupport Protocol 3 : Analysis of apparent on‐ and off‐kineticsSupport Protocol 4 : Analysis of changes in organelle overlap over time -
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Abstract Cross‐presentation was first observed serendipitously in the 1970s. The importance of it was quickly realized and subsequently attracted great attention from immunologists. Since then, our knowledge of the ability of certain antigen presenting cells to internalize, process, and load exogenous antigens onto MHC‐I molecules to cross‐prime CD8+T cells has increased significantly. Dendritic cells (DCs) are exceptional cross‐presenters, thus making them a great tool to study cross‐presentation but the relative rarity of DCs in circulation and in tissues makes it challenging to isolate sufficient numbers of cells to study this process in vitro. In this paper, we describe in detail two methods to culture DCs from bone‐marrow progenitors and a method to expand the numbers of DCs present in vivo as a source of endogenous bona‐fide cross‐presenting DCs. We also describe methods to assess cross‐presentation by DCs using the activation of primary CD8+T cells as a readout. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : Isolation of bone marrow progenitor cellsBasic Protocol 2 : In vitro differentiation of dendritic cells with GM‐CSFSupport Protocol 1 : Preparation of conditioned medium from GM‐CSF producing J558L cellsBasic Protocol 3 : In vitro differentiation of dendritic cells with Flt3LSupport Protocol 2 : Preparation of Flt3L containing medium from B16‐Flt3L cellsBasic Protocol 4 : Expansion of cDC1s in vivo for use in ex vivo experimentsBasic Protocol 5 : Characterizing resting and activated dendritic cellsBasic Protocol 6 : Dendritic cell stimulation, antigenic cargo, and fixationSupport Protocol 3 : Preparation of model antigen coated microbeadsSupport Protocol 4 : Preparation of apoptotic cellsSupport Protocol 5 : Preparation of recombinant bacteriaBasic Protocol 7 : Immunocytochemistry immunofluorescence (ICC/IF)Support Protocol 6 : Preparation of Alcian blue‐coated coverslipsBasic Protocol 8 : CD8+T cell activation to assess cross‐presentationSupport Protocol 7 : Isolation and labeling of CD8+T cells with CFSE -
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Abstract The heterogeneous injury pathophysiology of traumatic brain injury (TBI) is a barrier to developing highly sensitive and specific diagnostic tools. Phage display, a protein‒protein screening technique routinely used in drug development, has the potential to be a powerful biomarker discovery tool for TBI. However, analysis of these large and diverse phage libraries is a bottleneck to moving through the discovery pipeline in a timely and efficient manner. This article describes a unique discovery pipeline involving domain antibody (dAb) phage in vivo biopanning and next‐generation sequencing (NGS) analysis to identify targeting motifs that recognize distinct aspects of TBI pathology. To demonstrate this process, we conduct in vivo biopanning on the controlled cortical impact mouse model of experimental TBI at 1 and 7 days postinjury. Phage accumulation in target tissues is quantified via titers before NGS preparation and analysis. This phage display biomarker discovery pipeline for TBI successfully achieves discovery of temporally specific TBI targeting motifs and may further TBI biomarker research for other characteristics of injury. © 2021 Wiley Periodicals LLC.
This article was corrected on 19 July 2022. See the end of the full text for details.
Basic Protocol 1 : Phage production and purificationSupport Protocol : Controlled cortical impact modelBasic Protocol 2 : Injection and elution of phageBasic Protocol 3 : Amplicon sequencing and sequence analysis -
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Abstract Accurately mapping changes in cellular membrane potential across large groups of neurons is crucial for understanding the organization and maintenance of neural circuits. Measuring cellular voltage changes by optical means allows greater spatial resolution than traditional electrophysiology methods and is adaptable to high‐throughput imaging experiments. VoltageFluors, a class of voltage‐sensitive dyes, have recently been used to optically study the spontaneous activity of many neurons simultaneously in dissociated culture. VoltageFluors are particularly useful for experiments investigating differences in excitability and connectivity between neurons at different stages of development and in different disease models. The protocols in this article describe general procedures for preparing dissociated cultures, imaging spontaneous activity in dissociated cultures with VoltageFluors, and analyzing optical spontaneous activity data. © 2021 Wiley Periodicals LLC.
This article was corrected on 20 July 2022. See the end of the full text for details.
Basic Protocol 1 : Preparation of dissociated rat hippocampal or cortical culturesAlternate Protocol : Preparation of microisland dissociated culturesBasic Protocol 2 : Imaging of spontaneous activity in dissociated cultures using voltage‐sensitive dyesBasic Protocol 3 : Analysis of spontaneous activity imaging data
