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In order to restore fertility in patients with preserved testicular tissue, and in patients cured of pediatric cancer in particular, different approaches of in vitro maturation of (pre)pubertal testicular tissue have emerged with the aim of reproducing in vitro the complete process of spermatogenesis. These approaches have continued to evolve over the last 50 years. The aim of in vitro spermatogenesis is to generate in vitro spermatozoa from isolated spermatogonial stem cells (SSCs) or intact fragments of testicular tissue. Isolated SSCs, obtained after enzymatic and/or mechanical dissociation of testicular tissue, can be grown in liquid medium (two-dimensional culture) or within a matrix (three-dimensional culture). The culture of testicular tissue fragments or organotypic culture, most often performed on agarose gel in a gas-liquid interphase, allows the interactions of SSCs to be preserved with their microenvironment. Gradually, different models are emerging that aim to mimic the physiological conditions of spermatogenesis, particularly its spatio-temporal organization. This research has led to the production of functional spermatozoa and viable and fertile offspring in mice. Translational research has been set up to transfer these technologies to human testicular tissue.