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Abstract

Organoids are three-dimensional clusters of stem cells that serve as valuable models for studying organ development and disease. However, their effectiveness is limited because they can form a necrotic core due to lack of nutrients and oxygen. This study looks at nature-inspired methods to improve organoid survival through vascularization and structural changes. First, we compared split organoids to intact ones. We found that splitting increases surface area and improves media flow, which helps reduce hypoxic stress and cell death. Bulk RNA sequencing showed that split organoids had lower expression of pro-apoptotic and stress-related genes. To further address diffusion issues, we explored using plant blood vessel structures by removing the cells from spinach leaves and embedding them in alginate-gelatin hydrogels. Perfusion tests with dye injection showed 64.3% central coverage, confirming that plant-based scaffolds can effectively transport nutrients. These findings suggest that adding vascular-like networks to organoid systems can greatly enhance viability, scalability, and biological relevance. This opens up new possibilities for personalized medicine, regenerative therapies, and modeling neurological diseases.

Keywords

Biology, Tissue Engineering, Organoid Vascularization, Decellularized Plant Scaffolds, Brain Organoids

Introduction

Organoids have become one of the most transformative tools in biomedical research because they allow scientists to study human development, disease progression, and drug responses without relying on human or animal subjects. Brain organoids, which are three-dimensional clusters of neural cells derived from stem cells, have been especially valuable for modeling neurological disorders. However, there is a fundamental limitation preventing organoids from reaching their full scientific potential: the formation of necrotic cores, caused by inadequate nutrient and oxygen delivery to interior cells.¹ Growth often ceases after a few months, with cells dying in the core due to the lack of vascularization.² Without a functional vascular system, nutrients cannot diffuse effectively, and oxygen cannot reach cells deep inside the organoid. This severely restricts long-term studies, increases experimental costs, and reduces the reliability of research outcomes.

Developing a strategy to prevent necrosis in organoids is therefore a critical scientific need. One specific research gap relates to how nutrients can be efficiently delivered to the center of an organoid without damaging the structure or compromising the biological model. Previous work has explored methods to artificially vascularize organoids through advanced engineering techniques. For example, vascularized human cortical organoids (vhCOs) have been shown to develop “blood–brain barrier characteristics, including an increase in the expression of tight junctions”.² Although these engineered models offer promising results, they require complex biotechnology equipment and highly specialized cell culture systems, making them inaccessible to many research environments.

An alternative approach has emerged through cross-kingdom biomimicry, in which plant tissues are used as scaffolds for growing human cells.² This demonstrated that the vascular network of a spinach leaf can be decellularized and used as a perfusable scaffold for cardiac tissue, noting that plant tissues possess “excellent water transport and retention, interconnected porosity, and preexisting vascular networks”.³ This discovery established the feasibility of using plant vasculature as a low-cost, structurally robust platform for tissue engineering. If plant vascular systems can support fluid flow for cardiac cells, they may also provide a pathway to deliver nutrients and oxygen to brain organoids.

Recent work framing the neurovascular unit in organoids has emphasized the importance of sustained oxygen and nutrient delivery to reduce hypoxia-driven necrosis in organoid cores.⁴ Although vascularized human cortical organoids demonstrate improved viability and blood-brain barrier characteristics, these systems typically depend on specialized co-culture systems, perfusion hardware, and advanced microfabrication techniques.² In contrast, decellularized plant scaffolds provide naturally occurring perfusable conduits with low material costs and strong structural integrity.⁵ This creates an opportunity to evaluate whether plant-derived vascular systems may serve as scalable and accessible biomimetic alternatives for nutrient delivery within organoid culture systems.⁶

The research question guiding this investigation was: How effective is a plant-derived vascular scaffold in sustaining growth and improving nutrient delivery to brain organoids? We hypothesized that increasing nutrient accessibility through structural modifications, including organoid splitting and decellularized plant vasculature, would improve viability and reduce diffusion-related stress compared to traditional static culture conditions. Specifically, we predicted that split organoids would maintain high viability due to increased surface area exposure and that decellularized spinach vasculature would demonstrate measurable central perfusion throughout preserved vascular channels.

This study contributes both theoretical and practical significance to the field of organoid engineering. Theoretically, it investigates the feasibility of cross-kingdom vascular compatibility for nutrient transport in organoid systems. Practically, it evaluates a low-cost and accessible approach that may reduce organoid necrosis, decrease material waste, and improve the reliability of long-term experiments. The major findings of this study demonstrated that split organoids maintained viability above 98% and that decellularized spinach scaffolds preserved vascular integrity while achieving 64.3% perfusion coverage. Together, these findings support the potential of plant-derived vascular systems as scalable biomimetic platforms for future organoid culture applications.

Methods

Human Cortical Organoids

Human induced pluripotent stem cell (hiPSC)-derived cortical organoids were generated from the C4.1 female donor hiPSC line, which was previously established from peripheral blood obtained from a healthy adult donor and is not commercially available. All organoids used in this study originated from the same differentiation batch and were maintained in culture for at least 150 days in vitro (DIV) prior to experimentation. Cortical organoids were generated following existing protocols.⁷

Cortical organoids were generated using a previously established hiPSC differentiation protocol. Briefly, hiPSCs were expanded in Essential 8 medium on vitronectin-coated plates until reaching 80–90% confluence. Cells were dissociated into a single-cell suspension using Accutase, counted, and seeded into AggreWell™ 800 plates in Essential 8 medium supplemented with the ROCK inhibitor Y-27632 to generate uniform embryoid bodies. After 24 hours, embryoid bodies were transferred to ultra-low attachment plates and cultured in Essential 6 medium containing dorsomorphin and SB431542 to induce neural fate. Beginning on day 6, organoids were maintained in Neurobasal-A-based medium supplemented with fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) to promote neural progenitor expansion. From days 26-44, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were added to support neuronal maturation. After day 45, organoids were maintained in Neurobasal-A-based medium without additional patterning factors and fed every 3-4 days until use.

Organoid Viability to Compare the Benefits of Vasculature Integration

Human brain organoids cultured at the Birey Lab within the Department of Human Genetics at Emory University from established human induced pluripotent stem cell (iPSC) lines were provided through a research collaboration for experimental analysis. No patient-derived primary cells or embryonic stem cells were used in this study. Organoid preparation involved dissociation of stem cell clusters, centrifugation to form spherical organoids, plating organoids into standard culture wells, and dividing a subset of organoids into halves to increase surface exposure.

Five organoids were included in each experimental subset. In the control condition, organoids remained intact, while in the split condition organoids were divided under a microscope using a sterile scalpel and both halves were transferred into the same culture well. ROCK inhibitor (Y-27632) was added following dissociation to reduce apoptosis associated with cellular detachment and recombination. Organoids were maintained in organoid growth medium over a seven-day period before viability analysis was performed using Trypan Blue staining and automated cell counting.

Bulk RNA Sequencing for Molecular Analysis

Total RNA obtained from the previous extraction phase was processed for bulk sequencing using a spin column-based purification method. Twenty-five microliters of RNA sample was transferred into a spin column and subjected to sequential filtering involving lytic, purifying, and washing buffers followed by centrifugation and a dry spin to remove residual liquid. After elution, RNA concentration and purity were assessed using a Nanodrop spectrophotometer. One microliter of eluted RNA was analyzed spectrophotometrically and the resulting purity ratios were recorded for downstream applications.

Vasculature Biomimicry

Fresh spinach leaves were selected because they contain a highly branched, centrally converging vascular network structurally similar to mammalian capillary systems. Circular cutouts measuring approximately 5–10 mm in diameter were taken from the central region of each leaf using a scalpel. Samples were mounted on microscope slides and analyzed using light microscopy. Vein spacing, density, and structural integrity were quantified using ImageJ software.

A modified Franklin’s solution method was used for decellularization. A 1:1 solution of hydrogen peroxide and glacial acetic acid was prepared. Spinach leaves were boiled in water for 20–30 minutes before being submerged in Franklin’s solution for 1–2 hours until translucent. Leaves requiring additional clarification were briefly soaked in diluted bleach before rinsing and drying between filter paper.

A stabilizing hydrogel was prepared using sodium alginate, gelatin, and distilled water. Leaves were embedded in the hydrogel prior to dye perfusion testing. Food coloring was injected through the stem of each decellularized leaf using a syringe fitted with a 0.5–1 mm tip. Perfusion spread, leakage, and vascular coverage were recorded and quantified using ImageJ analysis.

Results and Discussion

This study investigated whether bioinspired vascular strategies could improve nutrient accessibility and reduce conditions associated with necrotic stress in brain organoids. The results demonstrated that both structural modification of organoids and plant-derived vascular scaffolds may support improved nutrient distribution without compromising viability.

The split organoid condition maintained viability above 98%, supporting the hypothesis that increasing surface area exposure can improve media accessibility while preserving cell survival. Although total cell density differed between conditions, both groups maintained high live-cell percentages throughout the experiment. These findings are consistent with previous studies suggesting that diffusion limitations contribute to hypoxia and necrotic core formation in dense organoid systems.⁴

The decellularized spinach scaffolds successfully preserved vascular architecture and demonstrated measurable fluid perfusion throughout central vascular channels. The observed 64.3% perfusion coverage indicates that plant-derived vascular systems can support fluid transport through structurally intact branching networks. These findings support previous research demonstrating the feasibility of plant vasculature as a biomimetic scaffold for tissue engineering applications.⁵

This study has several limitations. Dye perfusion was used as a proxy for nutrient transport and does not fully replicate physiological oxygen or metabolite diffusion. In addition, the sample size was limited, and experiments were performed using older organoids, which may affect generalizability to earlier developmental stages. Continuous perfusion systems and direct molecular validation of stress-response pathways were also outside the scope of the present study.

Future research should evaluate continuous-flow perfusion systems, investigate earlier-stage organoids, and complete transcriptomic analyses to determine whether improved nutrient accessibility correlates with reduced expression of stress-associated genes such as CASP3, DDIT3, and HIF1A. Additional work should also focus on improving scaffold reproducibility and optimizing vascular perfusion conditions.

In summary, the findings of this study suggest that bioinspired vascular scaffolds may provide an accessible and scalable approach for improving nutrient delivery within organoid culture systems. Decellularized plant vasculature demonstrated preserved structural integrity and measurable perfusion, supporting its potential application in future organoid engineering research.

Organoid Viability to Compare the Benefits of Vasculature Integration

Both control (N) and split (S) organoids maintained high viability above 98% across all replicates (Table 1). Control organoids demonstrated consistently higher overall cell density values, while split organoids maintained comparable live-cell percentages. Mean live-cell density measurements remained similar between conditions with minimal variation across replicates. Figure 1 illustrates the comparison of total and live-cell densities between control and split organoid conditions.

Table 1. Cell density (cells/mL), live cell density (cells/mL), and percent viability of control (Normal) and spinach scaffold (Split) organoids measured after culture. Data were collected from n = 5 control replicates and n = 6 split replicates. Live cell density was determined using automated cell counting. No statistical analysis was performed. N = Normal (control); S = Split (spinach scaffold)

Condition	Replicate	Cell Density (cells/mL)	Live Cell Density (cells/mL)	Live %
Normal	1	2,960,000	2,960,000	100
Normal	2	910,000	895,000	98
Normal	3	2,580,000	2,560,000	99
Normal	4	1,700,000	1,680,000	99
Normal	5	351,000	345,000	99
Split	1	4,350,000	4,330,000	100
Split	2	984,000	978,000	99
Split	3	2,750,000	2,740,000	100
Split	4	1,080,000	1,060,000	98
Split	5	1,230,000	1,220,000	99
Split	6	2,760,000	2,730,000	99

Figure 1 Caption: The title is “Comparison of Split vs. Control Organoids Live and Dead Cell Densities”. Bar graphs show the mean ± standard deviation (SD) of total cell density and live cell density for Normal (N) and Split (S) organoids, calculated from the data in Table 1. Error bars represent SD across biological replicates (n = 5 for Normal; n = 6 for Split). Cell viability remained above 98% in both groups, indicating minimal cell death. No statistical significance testing was performed. Graphs generated with the assistance of GitHub Copilot.

Bulk RNA Sequencing for Molecular Analysis

Nanodrop UV-Vis spectroscopy confirmed detectable RNA concentrations in both control and split organoid samples (Table 2). Absorbance spectra demonstrated peaks near 260 nm consistent with nucleic acid detection. A260/A280 and A260/A230 purity ratios varied across samples, with several normalized samples demonstrating lower concentration values following purification.

Table 2. RNA concentration (ng/µL) and purity ratios (A260/A280 and A260/A230) measured using NanoDrop UV-Vis spectrophotometry for RNA extracted from Normal (N) and Split (S) organoids. Samples 2 and 4 from the Split group were remeasured after normalization. Data represent individual biological replicates (n = 5 Normal; n = 6 Split). A260/A280 indicates protein contamination, and A260/A230 indicates salt or organic compound contamination. No statistical analysis was performed.

Condition	Sample	Concentration (ng/uL)	A260/A280	A260/A230
Normal	1	21.7	1.76	0.44
Normal	2	8.6	1.63	0.2
Normal	3	14.4	1.9	0.09
Normal	5	17	1.71	0.07
Normal	6	11.9	1.61	0.59
Split	1	22.5	1.88	0.61
Split	2	89.8	1.51	0.54
Split	3	21.7	1.88	0.75
Split	4	39.5	1.52	0.38
Split	6	23.7	1.82	0.18
Split	2 (Normalized)	2.9	1.59	0.26
Split	4 (Normalized)	5.2	1.5	0.17

Vasculature Biomimicry

Spinach leaves became translucent following decellularization while maintaining visible vascular architecture (Figure 2). Major and minor vascular channels remained structurally intact after treatment. Dye perfusion experiments demonstrated fluid movement throughout the vascular network. ImageJ analysis measured a total central area of 265,856 pixels and a dyed central area of 170,964 pixels, corresponding to a perfusion coverage of 64.3% (Figure 3).

Figure 2. Photograph showing the hydrogel injection procedure used to seed the decellularized spinach leaf scaffold through the leaf vasculature. Before the final injection shown, the needle insertion technique was practiced on two additional leaves to improve consistency and minimize damage to the scaffold. The image represents the successful injection used in the experiment.

Figure 3. Representative ImageJ analysis used to evaluate hydrogel distribution within the decellularized spinach leaf scaffold after injection. The software was used to visualize and assess scaffold coverage qualitatively. The image shown is representative of the successfully injected scaffold. No statistical analysis was performed.

Conclusion

This study examined two complementary, low-cost strategies for improving nutrient delivery to the interior of brain organoids: structural splitting to increase surface area, and decellularized plant scaffolds to provide a preformed vascular architecture. Split organoids maintained viability above 98%, supporting the hypothesis that increased surface area exposure reduces diffusion-related stress without compromising cell survival. Decellularized spinach leaves retained their branching vascular architecture after treatment and achieved 64.3% central perfusion coverage in dye-injection testing, demonstrating that plant-derived scaffolds can transport fluid through structurally intact channels. Together, these findings support the feasibility of cross-kingdom, biomimetic approaches to organoid vascularization as an accessible alternative to more complex bioengineering methods. Future work incorporating continuous-flow perfusion, earlier-stage organoids, and transcriptomic validation of stress-response genes will help clarify whether improved nutrient accessibility translates into measurable reductions in hypoxic and apoptotic signaling within the organoid core.

AI Acknowledgement

During the preparation of the work, the author used GitHub Copilot to generate the Figure 1 bar graphs. After using this tool, the author reviewed and edited the results accordingly and takes full responsibility for the content of the publication.

Acknowledgements

The author thanks Scott Robinson for mentorship throughout this project, and the Birey Lab, specifically Arvin Sarkissian, the Department of Human Genetics at Emory University for providing the human brain organoid samples used in this study.

References

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Authors

Aarushi Dhawan is a senior at FCS Innovation Academy High School in Alpharetta, Georgia. Her research interests include biomedical engineering and tissue engineering, with a focus on developing accessible, low-cost ways of improving organoid viability for rare disease modeling.