A Simple Temperature‐Controlled Method for Preserving Viability in Human Pluripotent Stem Cell‐Derived Retinal Organoids During Shipment

ABSTRACT

Retinal organoids derived from human induced pluripotent stem cells (hiPSCs) hold significant promise as a tool for advancing our understanding of human retinal development and disease, as well as for the development of therapeutic strategies. However, research utilizing hiPSC‐derived retinal organoid models has been constrained by logistical challenges. A notable issue is the increasing need for scientific collaboration, as experiments requiring coordination across multiple institutions often rely on the shipping of live tissues. Transporting retinal organoids over long distances without adequate climate control can lead to tissue degradation, which may interfere with histopathological analyses and impact the results of drug screening assays. Retinal organoids are most often shipped either without temperature regulation or with lithium‐ion battery‐powered ambient environment systems, which are heavily regulated by standard shipping companies, thus raising costs and introducing further logistical complications. In this study, we developed and evaluated a straightforward, cost‐effective, and dependable temperature‐controlled technology for shipping retinal organoids that can maintain adequate shipping temperature in colder months and decrease temperature fluctuations during shipment year‐round, preserving organoid tissue structure and viability.

A lithium‐free temperature‐controlled shipping system (YOTI) stabilizes physiologic temperatures during overnight transport of mature human iPSC‐derived retinal organoids. Compared to conventional ambient shipping, YOTI reduces temperature fluctuations, preserves retinal lamination, decreases cell death, and maintains rod photoreceptor marker expression. This accessible, cost‐effective method facilitates reliable inter‐institutional collaboration and reproducible organoid‐based research.

1. Introduction

The retina is a delicate and complex neural structure specialized in light detection and processing. As an extension of the central nervous system, the human retina lacks the ability to regenerate, meaning that any damage to retinal neurons—whether from trauma, infection, or degenerative disease—results in irreversible vision loss or blindness. This limitation has prompted researchers worldwide to explore the potential of human pluripotent stem cell‐derived retinal organoids (ROs) for translational research applications. ROs are three‐dimensional retinas cultured in vitro that closely mimic the cellular composition and organization of the native human retina [1, 2, 3, 4]. They can be generated from human embryonic stem cells or from patient‐derived induced pluripotent stem cells (hiPSC), and they can achieve a degree of functional maturation that allows them to respond to light stimulation [4, 5, 6, 7]. These attributes make ROs a powerful tool for modeling retinal diseases, testing potential therapeutic drugs, and developing cellular products for transplantation aimed at restoring vision or preventing vision loss [8].

Due to the increasing complexity of scientific research and technology, there is a growing need for multi‐institutional collaboration to fully leverage organoid technologies and advance potential therapeutic strategies. One significant challenge associated with these collaborations is the frequent requirement for long‐distance shipping of live ROs. To reduce variability and improve outcomes, it is essential to maintain tissue integrity during transport, ensuring that ROs remain suitable for further studies, which may include drug testing, histopathological evaluation, single‐cell RNA sequencing, and electrophysiological assessments.

Retinal organoids are typically shipped overnight at room temperature in cell culture flasks nestled inside Styrofoam containers, using commercially available controlled ambient environment systems, or employing costly shipping services [9, 10, 11]. While conventional shipping methods utilizing Styrofoam containers are convenient and cost‐effective, they are ineffective at maintaining a stable temperature, particularly when external environmental conditions fluctuate during transport, such as in aircraft cargo holds or delivery vehicles during colder months. In contrast, commercially available controlled ambient environment systems address these temperature challenges by employing heating devices powered by lithium‐ion batteries, ensuring a consistent internal temperature. However, these systems are costly and introduce significant logistical complexities when shipping through expedited delivery services like FedEx, DHL, and UPS, due to the regulatory requirements associated with the shipment of packages containing lithium batteries in aircraft cargo compartments. In this context, we investigated a more economical and logistically simple alternative method for shipping retinal organoids, focusing on preserving tissue viability throughout transit. Our results indicate that our shipping method controls temperature better than ambient shipping in a Styrofoam box, and that controlling temperature fluctuations leads to improved histology and decreased cell death.

2. Methods

2.1. Cell Lines and Retinal Organoid Generation

We used two human iPSC lines to generate retinal organoids in this study: A18945 (female, cord blood‐derived; obtained from Gibco) and iLC42‐3 (male, urine‐derived; generated at the University of Colorado). hiPSC lines were seeded on growth factor‐reduced Matrigel (Corning, 354,230), maintained in mTeSR1 medium (StemCell Technologies), and passaged at 60% to 70% confluence using dispase. They were routinely karyotyped and tested for mycoplasma.

Retinal organoid generation was performed as previously described [3, 4, 12]. The use of hiPSCs in this study conforms to the University of Colorado Institutional Biosafety Committee standards.

2.2. Incubator Setup and Environmental Tests

A commercially available 30‐oz, vacuum‐insulated YETI Rambler Travel Mug with a magnetic slider lid was used as the foundation for the development of a travel incubation system aimed at providing a simple, robust, cost‐effective, and repeatable method of shipping retinal organoids that maintains tissue viability. This innovation, referred to as the ‘YOTI’ (Your Organoid Temperature Incubator), includes a custom 3D‐printed partition insert designed to securely hold a cell culture flask containing the organoids, in addition to a series of heating packs and temperature probes that stabilize and monitor the environmental conditions during testing (Figure 1).

FIGURE 1.

The “Your Organoid Temperature Incubator”. YOTI configuration for shipping retinal organoids. Set up includes a custom 3D‐printed insert placed into a commercially available 30‐oz YETI Rambler Travel Mug, alongside heating packs and temperature/humidity probes. The whole system is housed within a Styrofoam container for overnight shipping via FedEx.

An Elitech GSP‐6G Digital Temperature Humidity Data Logger, with two detachable buffer probes, was used to monitor conditions both inside the YOTI and the ambient temperature and humidity in the packing crate. Interior temperature regulation of the YOTI was achieved by incorporating a UniHeat 40+ hour chemical heating pack, which was originally developed for the shipment of small pets (American Pioneer International LLC). The heating pack used has a peak surface temperature of 46°C, with a maintained temperature of 38°C. The custom insert not only secures the organoids, but also accommodates the probes and heating pack within the system. Costs for the YOTI set up total approximately $140 (excluding the cost of a 3D printer) at the time of this publication—YETI Rambler Travel Mug ~$40, Uniheat packs ~$30 (12 pack), data logger ~$60, resin for insert ~$10. An open access .stl file for 3D printing of the custom insert was deposited in https://github.com/ACS0021/3D‐prints. This setup was tested under three distinct environmental conditions: a freezer at −12°C, a refrigerator at 4°C, and at room temperature (~22°C), for a minimum of 24 h. Daily fluctuations in laboratory ambient temperature levels resulted in subtly different starting temperatures, and any instantaneous increase in heat at the start of an experiment was attributed to an artifact of heat transfer from the experimenter during set‐up.

2.3. Assessment of YOTI Performance Under Shipping Conditions

After evaluation, the most stable configuration was utilized for shipping retinal organoids from Aurora, Colorado, to Akron, Ohio, via standard overnight FedEx shipping. The YOTI configuration was placed inside a lidded Styrofoam box, which was then housed within a plastic shipping crate. During the colder months (December, January, February, March and April), the chemical heating pad (Uniheat small pets shipping warmer, American Pioneer International LLC.) was incorporated into the setup to provide warmth, ensuring that the organoids remained within the optimal temperature range. In contrast, during the warmer months (May, June, August, and October), the configuration successfully maintained the desired temperature without the heating pad, which was omitted from shipments during that period. Twenty shipments were performed in total.

2.4. Tissue Collection and Processing

ROs were imaged under bright field phase contrast microscopy with a 20X objective to assess RO quality and viability prior to shipping. ROs were placed in culture flasks with 70 mL BrainPhys media (Stem Cell Technologies) supplemented with 1× NEAA, 1× N2 (Invitrogen), 1.25 × GlutaMAX and 100 mM taurine. Flasks were placed inside the YOTI or in the Styrofoam container within the shipping box without temperature control (ambient condition). Upon arrival, ROs were imaged under bright field microscopy and subsequently fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature (RT). Control ROs were fixed by the same method without shipping. Samples were then washed 3 times in 1X PBS and subjected to a sucrose gradient (6.12%, 12.5%, and 25% sucrose in PBS) at 4°C until they reached osmotic equilibrium. ROs were embedded in 7.5% gelatin, 15% sucrose and flash frozen in dry ice. 10 μm‐thick sections were obtained using a Leica cryostat and processed for immunofluorescence or stored at −80°C until used.

2.5. Immunofluorescence Staining and TUNEL

Slides were dried at room temperature for 1 h, pap‐penned, and washed three times in PBS for 5 min. For TUNEL staining, slides were washed in 0.25% Triton X‐100 in PBS (PBST) for 15 min followed by a de‐ionized water wash, and they were then incubated in 1× Terminal Transferase (TdT) buffer (buffer 4 (NEB) 1:10 and CoCl₂ 1:10), at room temperature for 10 min in a humid chamber. After draining the solution, sections were incubated with TdT buffer 1:10, biotinylated‐dUTP 1:25, and TdT enzyme 1:100 for 1 h at 37°C in a humid chamber. Slides were then washed in 2X SSC for 15 min followed by a PBS wash, and blocked using 2% BSA in PBS for 10 min at room temperature. After washing, slides were incubated for 45 min in rhodamine avidin‐D (CAT: 2684302) 1:100, and washed in 0.25% PBST. For immunofluorescent staining, slides were blocked for 1 h in 10% normal donkey serum diluted in 0.25% PBST at RT, and incubated overnight at 4°C in primary antibody solution diluted in 2% normal donkey serum with 0.25% PBST. Primary antibodies used were NRL (CAT#:AF2945; R&D Systems) 1:100, Rhodopsin (CAT#: AB98887; Abcam) 1:100, PKCα (CAT#:21991–1‐AP; Proteintech) 1:250 and AP2α (3B5; DSHB). Slides were then washed PBS three times for 5 min and incubated for 1 h at RT in the corresponding secondary antibodies diluted 1:1000 in 2% normal donkey serum with 0.25% PBST. Secondary antibodies used were donkey‐anti‐mouse Alexafluor 546 (Invitrogen, cat# A10036), donkey‐anti‐goat Alexafluor 488 (Jackson Immunoresearch Laboratories; cat# 705–545‐003) and donkey‐anti‐rabbit Alexafluor 488 (Invitrogen, cat# A‐21206). Slides were washed in PBST for 5 min, followed by two 10 min washes in PBS, and incubated in DAPI solution for 10 min for nuclear counterstaining. Slides were then mounted in Fluoromount mounting medium, coverslipped, and stored at 4°C until imaging.

2.6. Image Analysis and Quantification

Images were acquired using a Nikon C2 Confocal Microscope (Minato City, Tokyo, Japan). The same acquisition parameters were used for all images. Images were stitched together using Nikon Elements Software and analyzed with Fiji software [13] (Bethesda, MD, USA). A minimum of five organoids per condition and per shipment were analyzed for each marker. Histology results from two independent shipments were pooled together for data analysis. For tissue architecture, the total length of the perimeter of the RO and the total length of organoid tissue that had normal lamination (outer nuclear layer with present or developing outer plexiform layer without photoreceptor rosettes, RPE tissue, or disorganized cells) was measured using Fiji Software (segmented line selection tool) and expressed as proportion of normally organized tissue. One section per RO, measured at its center, was quantified. For TUNEL staining, the number of labeled cells was manually counted and the area of retina sampled was quantified. Regions of organoids with disorganized tissue were excluded from this analysis. For immunofluorescence quantification of PKCα and AP2α, the relative area of positive immunostaining was calculated as a percentage of total retinal area estimated using DAPI staining (labeled area/DAPI‐stained area). Briefly, using the polygon selection tool, the ROI was defined by outlining the retina. The area of DAPI labeling was measured by manually thresholding the DAPI labeling within the ROI and calculating the thresholded area. The immunolabeling was then manually thresholded and the area was calculated. The same thresholds for each fluorescence channel were used to batch‐process all images for the same experiment. For rhodopsin and NRL cell quantification, double‐labeled images were counterstained with DAPI and imaged as above. NRL positive nuclei were manually counted by placing a dot over each NRL positive nuclei in the image using the cell counter tool, and the number of NRL cells that were co‐labeled with rhodopsin was manually quantified by overlaying the dots on a merged image of NRL/rhodopsin and counting the number of rhodopsin positive cells with dots over their nuclei. Rhodopsin‐labeled cells that were not co‐labeled with NRL were also quantified. Images displayed in figures represent average examples for each condition. Sample numbers in statistical analyses and graphs correspond to independent organoids. Five to 11 independent ROs were evaluated per condition for all image analyses (actual numbers for each experiment are specified in the figure legends).

2.7. Statistical Analyses

Statistical analyses were performed using GraphPad Prism 10.1.0 Software. Means of the groups were compared with Brown‐Forsythe Anova test and unpaired t‐tests with Welch's correction were utilized for post hoc testing. A p‐value < 0.05 was considered statistically significant. The False Discovery Rate (method of Benjamini, Krieger and Yakutiele) was used to correct for multiple comparisons. Bar graphs indicate the mean ± standard error of the mean (SEM), and individual values are plotted. Mean between shipments on each measure were also compared using Brown‐Forsyth Anova test and were non‐significant, so the data from two shipments were pooled. Variance between mean temperature data was analyzed using Levene's test, without correction. Data were split into multiple groups for analysis—warm and cold months for both ambient temperatures and YOTI temperatures, along with cold YOTI/warm ambient, and warm YOTI/cold ambient, with the unit of replication being the individual shipments. Cold months were defined as December, January, February, March and April, while warm months spanned May, June, August and October, with significance set at 0.05. Individual shipment data was analyzed for variability between YOTI and ambient conditions by generating frequency histograms of temperature data and testing for differences using Kolmogorov–Smirnov test with significance set at p < 0.05.

3. Results

3.1. Performance of the YOTI System Under Controlled and Real‐World Shipping Conditions

To address the need for a simple, reliable, and cost‐effective method for the transportation of ROs without compromising their viability, we evaluated a temperature‐controlled shipping system comprising a YETI Rambler Travel Mug, a custom‐designed 3D‐printed partition insert, and a series of heating packs and temperature probes, collectively referred to as the ‘YOTI’ (Your Organoid Temperature Incubator) (Figure 1). This system was engineered to sustain a stable internal temperature within physiologically relevant ranges.

To assess its efficacy, the YOTI was subjected to three distinct temperature environments: a standard freezer set at −12°C, a conventional refrigerator maintained at 4°C, and a laboratory countertop at room temperature (22°C). These conditions were selected to simulate the various temperatures encountered during potential shipping scenarios. The customized YOTI configuration successfully maintained temperatures within the range of 25°C–37°C for a duration of up to 20 h under below‐freezing, refrigerated, and room temperature conditions, compared to “ambient” conditions (which herein refer to temperature probes placed within the Styrofoam container but outside the YOTI) (Figure 2).

FIGURE 2.

YOTI performance on environmental control trials. YOTI configurations were optimized and trialed in various control environments. Specifically, the YOTI system, contained within a Styrofoam container normally used for shipping, was placed inside a −12°C freezer or a refrigerator at 4°C, or stored at room temperature (average 22°C). Temperatures within the YOTI and in the Styrofoam box (“ambient”) were monitored for up to 20 h. Blue lines represent averaged YOTI data, olive green lines indicate averaged ambient data, and the dashed line marks 37°C temperature. Sample sizes: N = 8 independent experiments for ambient; n = 9 independent experiments for YOTI.

To evaluate the performance of the system under real shipping conditions, ROs were shipped from Aurora, Colorado to Akron, Ohio, multiple times over the course of a year. The organoids were housed within the YOTI placed inside a Styrofoam container, and this assembly was enclosed within a plastic shipping crate secured with cable ties (Figure 1). The packages were shipped via FedEx Standard Overnight service, and the ROs were packaged within a couple of hours of pick‐up. Temperature data were collected across both cold months (November through April) and warmer months (May through October). A second set of ROs was shipped alongside the YOTI in the same Styrofoam container (“ambient”), and temperature probes were placed both inside the YOTI and inside the Styrofoam container, the latter mimicking a regular shipment of ROs without the YOTI. An additional set of organoids was retained and fixed for histological analysis (referred to as “control” or non‐shipped condition). Violin plots of temperature data were generated for cold months (Figure 3a) and warm months (Figure 3b). The average monthly temperatures for shipments from both locations were also compared for the ambient and YOTI temperature probes (Figure 3c). Results show that during the colder months, when the YOTI configuration included a chemical heat pack, the internal temperature of the YOTI was stabilized, and this configuration successfully maintained the ROs at physiological temperatures while ambient conditions did not. In these shipments, the average temperature inside the YOTI was 30.5°C, while the temperature outside the YOTI, within the Styrofoam container, was significantly lower, at 15.6°C (Figure 3c,d; p < 0.001). Conversely, during the warmer months, the temperature mean was not significantly different between the YOTI and ambient conditions (Figure 3d).

FIGURE 3.

Year‐round testing of the YOTI configuration. Utilizing the YOTI method, ROs were shipped from Aurora, Colorado to Akron, Ohio. (a, b) Violin plots represent temperature ranges within the YOTI (violet) and in the Styrofoam container (green) monitored throughout the length of the shipping (Fedex overnight), on shipments sent at different times throughout a year. December, January, February, March and April were considered cold months in these regions and a heating pad was used (a). During warmer months (May, June, August and October), no heating pad was used (b). (c) The mean temperature of the shipments was plotted with lines representing the average of 13 shipping trials and ribbon representing standard error of the mean. Violet lines represent YOTI data and green lines indicate ambient data. (d) When analyzed using a Levene's test, the difference in the range of temperature variations between the ambient and YOTI conditions during the cold months was found to be statistically significant, with p = 0.001. Error bars represent SEM. Total shipments for each month: December—1, January—1, February—3, March—5, April—2 (cold month total = 12), May—3, June—2, August—1, October—2 (warm month total = 8).

3.2. Assessment of RO Tissue Structure and Viability After Shipment Using the YOTI System

ROs derived from hiPSC recapitulate the structural development of human retina, with the exception of a fovea [1, 2, 3, 4, 6, 14, 15]. Typically, neurogenesis, differentiation and lamination of ROs follow the time course of retinal development in vivo, although variability between and within ROs has been reported [2, 6, 14, 15, 16]. To study differences in retinal structure caused by shipping conditions, we performed a histological evaluation of aged ROs (220 days of differentiation) shipped in the YOTI and ROs shipped at ambient temperature conditions and compared them to control (not‐shipped) ROs. Individual temperature data for two shipments used in histological analysis were plotted (Figure S1). These shipments corresponded to cold (early March) and warm (August) months. While the cold shipment led to ambient temperatures below 0°C, YOTI temperatures were more stable (Figure S1a). Warm temperature shipment data showed cooler temperatures in the YOTI condition without a heat pack, but while mean temperatures in warm months were not significantly different between ambient and YOTI conditions, there were significant differences in temperature variability between these conditions. Indeed, when frequency histograms of temperature data were plotted using Prism (Figure S1c–f) and differences were evaluated by the Kolmogorov–Smirnov test, significant differences in temperature variability were observed in both warm and cold shipments (p < 0.01).

We then assessed the retinal structure of shipped and control ROs (Figure 4). Brightfield images were taken prior to (Figure 4a,b) and after shipping in the YOTI (Figure 4c), showing preservation of inner/outer segment structures (arrows). ROs were then fixed, and DAPI‐stained cryosections were analyzed for architectural integrity (Figure 4d–g). Of note, retinal organoids at this advanced age are expected to have an outer nuclear layer with maturing photoreceptors, the initial stages of outer plexiform layer formation, and inner nuclear layer neurons. However, the majority of retinal ganglion cells are lost in organoid models after approximately 100–120 days of differentiation, likely due to a lack of neurotrophic feedback from brain targets, and thus the inner plexiform layer does not form and there is a variable loss of amacrine interneurons [6, 17, 18]. In our study, we observed that the development of a clearly defined OPL varied between ROs as well as regionally within individual ROs; however, both control ROs and YOTI‐shipped ROs had more retinal tissue that remained properly organized than ambient‐shipped ROs, as defined by the appearance of an organized outer nuclear layer free of rosettes and with at least early stages of outer plexiform layer formation (Figure 4d–f). Quantitative image analysis confirmed a statistically significant loss of retinal tissue organization in ROs shipped at ambient temperature conditions compared to both control and YOTI‐shipped ROs (Figure 4g). Since all organoids were chosen based on a translucent, organized structure under brightfield microscopy before shipping and distributed randomly across conditions, this suggests that shipping in ambient conditions alters the structural integrity of ROs.

FIGURE 4.

Temperature control during shipping maintains retinal structure. (a–c) Live ROs at 220 days of differentiation were photographed under bright field illumination at 4× (a) and 20× (b) magnification prior to shipping, shipped using the YOTI configuration, and re‐imaged upon receipt at 20× magnification (c), using an inverted microscope. Arrows point at photoreceptor inner/outer segment structures, which are maintained after shipment. (d–f) ROs were fixed and tissue architecture was examined after DAPI nuclear staining. In confocal micrographs, control (not shipped) ROs show typical retinal lamination (d), YOTI shipped ROs (e) show similar lamination to controls, and ambient shipped ROs within the Styrofoam container (f) show structural abnormalities and decreased lamination. (g) Quantification of the proportion of retinal lamination shows significantly decreased lamination in ambient shipped ROs compared to both control and YOTI conditions. N = 11 ROs per condition, corresponding to the two shipments described in Figure S1. Brown–Forsythe Anova with Welch's t‐test, * p < 0.05, ***p < 0.001; different symbols represent ROs from 2 different shipments. Scale bars = 100 μm.

ROs shipped using the YOTI configuration displayed significantly lower levels of cell death than those shipped under ambient conditions (Figure 5). This was evidenced by the TUNEL assay, which showed that both control ROs (Figure 5a–c) and YOTI‐shipped ROs (Figure 5d–f) had fewer TUNEL (+) cells than ambient‐shipped ROs (Figure 5g–i). These differences were statistically significant (Figure 5j), suggesting that shipping with temperature control increases cell viability over ambient shipping conditions.

FIGURE 5.

Cell death is increased in Ambient shipping conditions. (a–i) Representative confocal micrographs show TUNEL and DAPI staining on cryosections of control ROs that were not shipped and maintained in normal culture conditions (CTR; a–c), ROs shipped in the YOTI configuration (d–f), and ROs shipped in the Styrofoam container (Ambient; g–i). (j) TUNEL quantification shows a significant increase in cell death in ambient shipped ROs compared to controls and YOTI shipped ROs. N = 8 RO per condition; Brown‐Forsythe Anova with Welch's t‐test, * p < 0.05; different symbols represent ROs from the 2 different shipments described in Figure S1. Scale bar = 50 μm.

3.3. Preservation of Cellular Composition Under YOTI Shipping Conditions

Immunolabeling and single‐cell RNA sequencing studies have shown that all major retinal cell types develop in ROs, and that RO cellular composition depends primarily on their developmental stage [6, 15]. Thus, to evaluate whether shipping conditions affect RO composition, we used immunofluorescence staining to compare some of the major retinal neuron types in shipped ROs at 220 days of differentiation (e.g., rod photoreceptors, rod bipolar cells and amacrine cells) between YOTI and ambient shipping conditions as well as with control, non‐shipped ROs. Amacrine cells, labeled with antibodies to AP2α (Figure 6a,b,e,f,i,j), and rod bipolar cells, labeled with antibodies to PKCα (Figure 6c,d,g,h,k,l), showed no significant differences across conditions (Figure 6m). To assess potential differences in rod photoreceptors, we then performed immunolabeling for NRL and found that, even though this marker was expressed in all conditions (Figure 7a,e,i) the number of NRL (+) cells was significantly decreased in ambient‐shipped ROs compared to controls (Figure 7m; p < 0.001). Conversely, the number of NRL (+) cells was intermediate in YOTI‐shipped ROs, but not significantly different from controls or ambient conditions (Figure 7m).

FIGURE 6.

Shipping does not impact the numbers of amacrine or bipolar cells. (a–l) ROs were maintained in normal conditions (CTR; a–d), shipped in the YOTI configuration (a‐h), or shipped in ambient conditions (i–l). ROs were fixed upon receipt, and cryosections were immunolabeled for AP2α, a marker of amacrine cells, or PKCα a marker of rod bipolar cells. Confocal micrographs show similar staining patterns across conditions. (m) Quantification of relative AP2α and PKCα labeled areas shows no significant differences among groups for either marker (N = 8 RO per condition; Brown‐Forsythe Anova with Welch's t‐test; different symbols represent ROs from the 2 different shipments described in Figure S1). Scale bar = 50 μm.

FIGURE 7.

YOTI shipment preserves rod photoreceptor markers better than ambient conditions. (a–l) Representative confocal micrographs show immunolabeling for rod photoreceptor markers rhodopsin (Rhod; green) and NRL (red) on ROs maintained under control conditions (CTR; a–d), or shipped using the YOTI (e–h) or ambient (i–l) configurations. Expression of both markers appears decreased in ambient shipping conditions (i–l). (m) Quantification of NRL (+) cells shows a significant difference between control and ambient conditions, while YOTI conditions show an intermediate outcome. (n) Quantification of rhodopsin (+) cells shows significant difference among all conditions, YOTI shipped ROs showing intermediate outcomes between the other two conditions. (o) A similar pattern was observed in the quantification of NRL(+) and rhodopsin (+) double‐labeled cells. N = 5–8 ROs per condition; Brown–Forsythe Anova with Welch's t‐test, ***p < 0.001 and **p < 0.01, *p < 0.05; different symbols represent ROs from the 2 different shipments described in Figure S1. Scale bar = 50 μm.

NRL expression marks rod cell fate and precedes expression of visual cycle proteins like rhodopsin [19]. Rhodopsin expression is activated later in maturing rods and is necessary for visual function [20, 21]. To determine if shipping ROs decreased rhodopsin expression, we quantified the number of rhodopsin‐positive cells under the different conditions. The number of rhodopsin immunolabeled cells was decreased in ambient conditions compared to control and YOTI conditions (Figure 7b,f,j). As with NRL, rhodopsin labeling in YOTI‐shipped ROs was intermediate between control and ambient conditions, but in this case, the difference in cell numbers was statistically significant (Figure 7n; p < 0.01 between ambient and YOTI; p < 0.05 between YOTI and control). Co‐labeling of NRL and rhodopsin showed similar results, with significant differences in double labeled cells seen between control and ambient conditions (Figure 7c,k,o; p < 0.001), between YOTI and ambient conditions (Figure 7g,k,o; p < 0.01), and between YOTI and controls (Figure 7c,g,o; p < 0.01). This may be because the number of rhodopsin‐expressing cells and NRL‐rhodopsin co‐labeled cells is nearly the same. Together, these results indicate that temperature control is an important factor in maintaining photoreceptor marker expression.

4. Discussion

Without temperature control, as is commonly the case for RO shipments between institutions, our experience and that of other colleagues indicate that ROs shipped overnight during winter months in North America occasionally arrive frozen, despite being contained in a Styrofoam box (personal communication with the authors). Thus, we have established a new, simple, and cost‐effective method for shipping ROs under temperature‐controlled conditions. The fundamental findings of this study are that the YOTI maintains temperature even under freezing ambient conditions and shows decreased temperature fluctuations during warmer months (Figures 1, 2, 3; Figure S1). Moreover, shipping ROs in the YOTI configuration leads to improved RO histology (Figure 4), decreased cell death (Figure 5), and increased numbers of photoreceptors (Figure 7) compared to ambient shipping conditions (with rhodopsin results intermediate between non‐shipped control and ambient shipping conditions), while the numbers of other retinal cell type markers remained similar across all conditions. Notably, while the mean temperature for warm shipments was similar between YOTI and ambient, temperature fluctuation was significantly different (Figure S1). Together, these results lead us to hypothesize that ROs do not tolerate temperature variations during shipment, necessitating temperature control innovations.

To illustrate the cost‐effective merits of the YOTI, when compared to the shipping set up used by Singh et al. [11], the YOTI configuration, excluding a 3D printer but including the resin for the insert, the YETI Rambler Travel Mug, heat packs and temperature probe totals around $140 at the time of publication. The Darwin Chambers incubator alone used by Singh and colleagues currently retails for ~$860, making the YOTI an elegant and highly cost‐effective method of shipping retinal organoids for research.

While chemical heat packs exhibit a non‐linear exothermic profile that begins with a peak followed by a gradual heat decline, the YOTI and surrounding Styrofoam container attenuate these fluctuations, as reflected in the relatively stable temperature traces observed across shipments. Although phase‐change materials and electronic temperature controllers offer greater precision, they introduce cost barriers or, in the case of lithium‐battery‐powered systems, regulatory restrictions on air shipment that limit accessibility for many research groups, the central problem this method is designed to address.

Our results align with those of Singh et al., whose study used a lithium battery‐containing incubator, reported temperatures ranging from 33°C to 34°C when shipping overnight from California to Michigan, and found good cell viability assessed by cleaved caspase 3 immunolabeling and Sytox green staining [11]. Our study expands on those observations by comparing the temperature range and fluctuation between our system and the ambient shipping method, and by comparing RO viability between those shipping conditions and with non‐shipped ROs, which has not been done before. Additionally, Singh et al. evaluated ROs shipped at early developmental stages, when the retinal neuroepithelium is composed primarily of progenitor cells [1, 5, 11, 14]. Our study, on the other hand, evaluates mature ROs with inner nuclear layer and photoreceptor cells to assess the impact of shipping on retinal neurons. Finally, the method we present is simple, cost‐effective, and does not rely on lithium batteries, allowing shipment by any air carrier.

Another study examined the effects of shipping ROs between England and Germany in a Stryofoam box, employing the services of a specialized shipping company, followed by incubation at 37°C for an additional 7 days before analysis [9]. This study found no difference between ROs shipped at ambient temperatures and non‐shipped ROs. However, methodological differences may account for the discrepancy in results. For example, the organoids in that study were shipped at Days 135 and 160 of differentiation, when neurogenesis is still occurring, and ROs were incubated for 7 more days after shipment prior to fixation. These conditions may allow more progenitor cells to differentiate, or maturation markers to recover. In our study, ROs were 220 days old, which is after most neurogenesis has occurred, and were fixed immediately upon arrival. Notably, more mature cells may be more susceptible to temperature fluctuations. Shipment testing with younger organoids could serve to confirm this hypothesis. Additionally, Georgiou et al. [9] provide temperature data for one shipment, but without information on weather conditions or time of the year, the performance variability under more extreme weather conditions cannot be determined.

Another interesting result from our study is that we saw differences in the density of NRL positive cells between non‐shipped controls and ambient conditions, but NRL positive cells were not significantly different between YOTI and ambient or YOTI and control, despite the fact that rhodopsin positive cells and rhodopsin and NRL co‐labeled cells were significantly different, with the YOTI configuration yielding intermediate results between ambient shipping and control non‐shipped conditions (Figure 7). We hypothesize that rhodopsin‐positive and co‐labeled cells may represent a subset of more mature rod photoreceptors with higher susceptibility to damage and death under ambient temperature conditions (due to temperature extremes or to temperature fluctuations), while NRL single‐labeled cells are more resilient (and ostensibly less mature). The increase in TUNEL positive cells in the ambient condition compared to the others may partially explain this result (Figure 5). However, it does not explain the difference in rhodopsin‐positive cells between control and YOTI conditions, which lacked a statistically significant difference in TUNEL positive cells. An alternative hypothesis may be that the mature rod marker rhodopsin may be lost upon stress conditions, while the definitive rod fate marker NRL is not. Further studies are needed to test these hypotheses.

One limitation of our study is that it does not address the physiology of shipped ROs, as no quantitative assays have yet been developed to allow comparative electrophysiological assessments in ROs at these advanced differentiation stages, when retinal ganglion cells are no longer present in meaningful proportions in ROs and therefore microarray technology cannot be used [7]. Future studies should examine the internal conditions of the YOTI during transit, including CO₂ levels, O₂ depletion, and media pH, to better characterize the physiological environment experienced by ROs during shipment and to determine whether temperature fluctuations or internal changes to the solution are the primary drivers of the structural and cellular changes we observed. Notably, the BrainPhys media used here is HEPES‐buffered, which partially mitigates pH sensitivity to CO₂ fluctuations compared to bicarbonate‐buffered formulations, though this does not fully preclude the possibility of metabolic gas accumulation over extended transit times. In addition to this, future work could also look at establishing datasets collected between different locations and climates, along with longer shipping durations and/or multi‐leg transit, as the work presented here looks at only one destination pairing. Additional limitations include a relatively low number of ROs analyzed, derived from two iPSC lines and evaluated at 220 days of differentiation. Thus, future studies should incorporate additional cell lines and organoid ages.

In summary, many research groups working on ROs are collaborating at a distance, and thus, it is important to develop and validate shipping methods that maintain the highest quality ROs. The YOTI system presented here provides an easy and affordable alternative for shipping ROs, with significant advantages for preserving organoid structural integrity and viability. The YOTI's benefit of temperature maintenance is most meaningful during cold weather months, since in warm months average temperatures can be similar to ambient, while the decrease in temperature fluctuations afforded by the YOTI may provide benefits year‐round. Our data suggest that ROs shipped with temperature control undergo less structural disorganization and cell death than those shipped in ambient conditions, particularly during cold‐season shipping. Further, our results also show that rhodopsin expression is decreased in ROs shipped at ambient conditions and that this outcome is partially ameliorated by shipping in temperature‐controlled conditions. The simple method presented here can be readily utilized by other researchers shipping ROs using any air carrier and could potentially be applied to organoids derived from other tissues.

Author Contributions

K.C., M.J.T., M.H., J.R.O., A.C.S.: performed experiments, data collection and data assembly. K.C., M.J.T., J.A.B., J.M.R., M.N.V.: conceptualization and design of experiments. K.C., M.J.T., J.M.R., M.N.V.: data analysis and interpretation. K.C., J.M.R., M.N.V.: manuscript draft writing. K.C., M.J.T., M.H., J.R.O., A.C.S., J.A.B., J.M.R., M.N.V.: manuscript editing. J.M.R., M.N.V.: supervision and project administration. J.M.R., M.N.V.: funding acquisition. All authors have approved the final version of the manuscript for publication.

Funding

This work was supported by NIH R21EY033127 to J.M.R. and M.N.V., by the CellSight Development Fund, and by an Unrestricted Grant to the Department of Ophthalmology at the University of Colorado from Research to Prevent Blindness.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Individual shipment temperature data. Temperature data from RO shipments at cold temperatures (a, c, e; March) and warm temperatures (b, d, f; August) from which histological data were collected. (a, b) Graphs of temperature recordings show that the difference in temperature fluctuations between ambient and YOTI conditions during transit was evident in both cold (a) and warm (b) months, even though the mean temperatures appeared different only in the former. (c, d) Frequency histograms of temperature data in the cold month shipment show significant variability between YOTI (c) and ambient (d) conditions, indicating higher temperature fluctuations in the latter. (e, f) The same result was observed in the warm month shipment, where frequency histograms also showed significant differences in temperature fluctuations between YOTI (e) and ambient (f) conditions. Komolgorov–Smirnov test was used to test for significance between temperature frequency data and was < 0.01 in both cold and warm shipments.

Data Availability Statement

An open access .stl file for 3D printing of the custom insert was deposited in https://github.com/ACS0021/3D‐prints. All additional data that support the findings of this study are available in the Materials and Methods, Results, and/or Supporting Information of this article.