**5. Limitations and deficiencies**

In recent years, although the technology of retinal organoids has made great progress, it is still beyond the reach of our existing tools and technologies to construct retinal organoids with the same structure and physiological functions as the mature retina in vivo. Our research on retinal organoids is still in its infancy and there are some limitations to overcome.

Long-term maintenance of retinal organoids depends on oxygen and nutrients, and in our existing culture system, oxygen diffusion limits the size of retinal organoids and the development of their internal cells, especially ganglion cells. Currently, we are trying to introduce a combination of tissue engineering techniques to solve this problem, such as the use of bioreactors and retinal chips [62, 63, 65]. The absence of vascular system also limits the long-term maintenance of organoids. Microglia are the resident immune cells of the central nervous system and are particularly important for the development of the retina, which can regulate the survival of neurons and prune synapses [141]. Co-differentiation of retinal organoids and vascular tissues or microglia in a dish is challenging because they come from different germ layers. The retina develops from the ectoderm, while vascular tissue-associated cells and microglia originate from the mesoderm. Therefore, we usually choose to achieve the complexity of retinal organoids through co-culture. In recent years, the realization of vascular structure in human brain has made some progress. After transplantation of human cerebral organs into the cerebral cortex of mice, the growth of blood vessels in mice was induced to increase the survival and maturation of cells [142]. In vitro, a study found that ectopic expression of human ETS variant 2 (ETV2) in hESCs can form a complex vascular-like network in human cortical organs and promote the maturation of organoid function [143]. In the future, we also expect that 3D printing of vascular tissue [144] and co-culture with mesodermal progenitor cells [145] will make the differentiation system of retinal organoids more perfect. For the retina in a petri dish to function, the most important point is to establish synaptic connections and form functional circuits. While our differentiated retina can form synaptic connections now, it's not nearly as good as the complex network of synapses in the retina in vivo. Even retinal organoids derived from normal stem cell lines respond poorly to light. This may be due to the gradual degeneration of ganglion cells during late differentiation and lack of connection to the brain, which hindrance our assessment of retinal functional circuits. It may also be associated with limited growth of the outer segment due to the lack of direct interaction with RPE. Retinal organoid technology has solved the problem of cell-cell interaction, but in organisms, tissue-to-tissue and organ-to-organ interactions remain important for development. For example, the relationship of the retina to the lens, ciliary body and cornea, and the relationship of the retina to the brain.

## **6. Conclusions**

It's an exciting time, and technological advances have made a lot of things possible. Retinal organoids are our research tools for overcoming retinal diseases. It allows us to further understand the development and maturation of the retina, reproduce disease pathology and phenotypes in vitro, and explore the feasibility of gene therapy. In addition, it provides us with cells for cell transplantation and drug screening. We have enjoyed the great benefits brought by retinal organoids. However, their defects

*Retinal Organoids over the Decade DOI: http://dx.doi.org/10.5772/intechopen.104258*

and deficiencies are also prominent, which is the direction we need to work towards. There is still a long way to go in the development of retinal organoids, and we expect that technological breakthroughs will enable us to advance to the next level in this field in the future.
