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Testes Fragment Cultures
In fragment cultures, the testis is removed and fragments of tissue are cultured in supplemental media containing different growth factors to induce spermatogenesis and form functional gametes. The development of this culture technique has taken place mainly with the use of animal models e.g. mice or rat testis tissue.

The advantage of using this method is that it maintains the natural spatial arrangement of the seminiferous tubules. However, hypoxia is a recurring problem in these cultures where the low oxygen supply hinders the development and maturation of spermatids (significantly more in adult than immature testis tissues). Other challenges with this type of culture include maintaining the structure of the seminiferous tubules which makes it more difficult for longer-term cell cultures as the tissue structures can flatten out making it hard to work with. To resolve some of these issues, 3D cultures can be used.

In 2012, mature spermatozoa capable of fertilization was isolated from in vitro culture of immature mouse testis tissue.

3D Cultures
3D cultures use sponge, models or scaffolds that resemble the elements of the extracellular matrix to achieve a more natural spatial structure of the seminiferous tubules and to better represent the tissues and the interaction between different cell types in an ex vivo experiment. Different components of the extracellular matrix such as collagen, agar and calcium alginate are commonly used to form the gel or scaffold which can provide oxygen and nutrients. To propagate 3D cultures, testicular cell cultures are imbedded into the porous sponge/scaffold and allowed to colonise the structure which can then survive for several weeks to allow spermatogonia to differentiate and mature into spermatozoa.

In addition, shaking 3D cultures during the seeding process allows for an increased oxygen supply which helps overcome the issue of hypoxia and so improves the lifespan of cells.

In contrast to monocultures, fragment/3D cultures are able to establish in vitro conditions that can somewhat resemble the testicular micro-environment to allow a more accurate study of the testicular physiology and its associations with the in vitro development of sperm cells.