Efficient transduction of pancreas tissue slices with genetically encoded calcium integrators

Reviewer #1 (Public review):

Summary:

The authors aimed to overcome a major technical limitation in pancreatic slice research - the inefficient viral transduction of dense, enzyme-active human pancreas tissue - while maintaining tissue integrity and physiological responsiveness. They developed a modified culture and infection protocol that incorporates gentle orbital agitation, removal of protease inhibitors, and physiological temperature during adenoviral transduction. This method increased transduction efficiency by approximately threefold without impairing insulin secretion or calcium signaling responses.

Strengths:

The study's major strengths are its clear methodological innovation, experiment optimization, and multiparametric validation. The authors provide compelling evidence that their approach enhances the expression of genetically encoded calcium indicators (GCaMP6m) and integrators (CaMPARI2), preserving both endocrine and exocrine cell functionality. The demonstration of targeted biosensor expression in β-cells and multiplexed imaging of redox and calcium dynamics highlights the versatility of the system. The CaMPARI2-based approach is particularly impactful, as it decouples maximum calcium response assessment from real-time imaging, thereby increasing throughput and reducing bias. The authors successfully apply the technique to samples from non-diabetic, T1D, and T2D donors, revealing disease-relevant alterations in β-cell calcium responses consistent with known physiological dysfunctions. The analysis of islet size versus calcium response further underscores the utility of this platform for probing structure-function relationships in situ.

Weaknesses:

The primary limitations are a lack of live/dead assessment to differentiate viability-related effects from methodological improvements, a lack of quantification of the transduction efficiency (while relative efficiency is clearly increased, it is not shown what is absolute efficiency is), lack of IF confirmation of the cell-specific transduction efficiency. These limitations, however, do not detract from the overall strength of the technical advance.

Overall, this work offers a convincing and practical advance for the diabetes and islet biology community. It substantially improves the toolkit available for live human pancreas studies and will likely catalyze further mechanistic investigations of islet heterogeneity, disease progression, and therapeutic response.

Reviewer #2 (Public review):

(1) The photoconversion protocol requires a more detailed and quantitative discussion. The current description ("5 s pulses for 5 min, leading to 2.5 min of total light delivery") is too brief to evaluate whether the chosen illumination parameters maintain the CaMPARI2 signal within its linear dynamic range. Because CaMPARI2 photoconversion reflects the time integral of 405 nm photoconverting light exposure in the presence of intracellular [Ca²⁺], the red/green fluorescence ratio is directly proportional to cumulative illumination time until saturation occurs. Previous characterization (PMID: 30361563) shows that photoconversion is approximately linear over the first 0-80 s of 405 nm exposure, after which red fluorescence plateaus. The total exposure used here (=150 s) may therefore exceed the linear regime, potentially obscuring differences between cells with moderate versus strong Ca²⁺ activity. The authors should (i) justify the selected illumination parameters, (ii) provide evidence that the chosen conditions remain within the linear response range for the specific optical setup, (iii) discuss how overexposure might affect quantitative interpretation of red/green ratios and comparisons between experimental groups. Inclusion of calibration data would substantially strengthen the methodological rigor and reproducibility of the study.

(2) For Figure 8a (middle panels), the data points for 16G and KCl show overlaps, raising the possibility that at it 16G may already be saturated. The authors should comment on the potential for CaMPARI2 saturation at 16G, and clarify whether this affects the interpretation of the KCl results "At maximal stimulation by KCl, there was no size-function correlation (R = 0.15, p = 0.14)."

(3) The term "calcium activity" is used throughout the manuscript but remains vague. Pancreatic islets typically display a biphasic Ca²⁺ response to high glucose-an initial sustained peak followed by repetitive oscillations - and these phases differ in both kinetics and physiological meaning. Ca²⁺ responses are usually quantified using parameters such as rise time, amplitude, and duration for the initial peak, and amplitude, frequency, burst duration, and duty cycle for the oscillatory phase. The authors should clarify how "calcium activity" is defined in their analyses and discuss the appropriateness of directly comparing Ca²⁺ signals with distinct temporal patterns.

(4) The CaMPARI2 red/green ratio reflects the time-integral of 405 nm photoconverting light exposure in the presence of Ca²⁺, two Ca²⁺ responses with the same duty cycle but different amplitudes could, in principle, yield the same red/green ratios. This raises an important question regarding how well the CaMPARI2 signal distinguishes differences in Ca²⁺ amplitude versus time spent above threshold. The authors should directly relate single-cell Ca²⁺ traces to corresponding red/green ratios to demonstrate the extent to which CaMPARI2 photoconversion truly reflects "Ca²⁺ activity." Such validation would clarify whether the metric is sensitive to variations in oscillation amplitude, duty cycle, or both, and would strengthen the interpretation of CaMPARI2-based functional comparisons.

Reviewer #3 (Public review):

Summary:

Lazimi and coworkers present an updated experimental protocol by which viral vectors can be used with live pancreas slices in order to efficiently transduce fluorescent protein biosensors. This is of high importance, given that live human pancreas slices provide a means to study islet function while maintaining the architecture of the local environment. Thus, efficiently delivering a wide range of fluorescent protein biosensors provides expanded capabilities to study the human islet and its dysfunction in type 1 and type 2 diabetes. The authors demonstrate the improved transduction provided by their revised protocol, which includes orbital culture, while retaining or, in some cases, improving cell viability, hormone release, and Ca2+ responses. Further, the authors demonstrate how a 'Ca2+ integrator', CAMPARI2, can be used to profile the Ca2+ response of large numbers of cells and islets, to capture the variability in islet responses in healthy and diabetic cases.

Strengths:

The data presented are generally robust, and the methods are well described, such that this protocol could be repeated by other investigators. All findings are representative of multiple donors. Importantly, the data is highly novel.

Weaknesses:

Weaknesses in the manuscript mainly include a lack of technical details by which data is presented or analyzed, as well as caveats by which certain data related to islet size are interpreted.