An integral component of the manufacture of a skin substitute is the cryopreservation of the complete skin construct. Under this demand, investigations were carried out in the present work in the case of cryopreservation of human fibroblasts and keratinocytes composed to organotypical skin substitutes (OTS). Two scaffolds made up of gelatine and collagen/elastin were seeded with human fibroblasts via centrifugation method. Subsequent human keratinocytes were applied on the preceded scaffolds and cultivated under air-exposed conditions. For the investigation of the cryopreservation, OTS were frozen after 10 days cultivation via computer-controlled CryoMed included defined freezing conditions. After 24 hours storage in fluid nitrogen the OTSs were thawed and recultivated under airlift conditions. After that metabolic activity and immunfluorescent staining was analyzed in comparison with conventionally produced OTSs on basis of collagengel and/or OTSs based on scaffolds without cryopreservation. It could be assessed that cryopreservation has no negative influence on vitality and differentiation capacity of the cultivated constructs. The determination of OTS vitality after 14 days airlift culture delivered persistent higher metabolic activities of the scaffold based constructs in comparison with the corresponding controls. This could be confirmed by investigation of OTSs with and without cryopreservation. All expression patterns of differentiation marker could be detected after cryopreservation and subsequent recultivation. The results from cryopreservation of OTSs introduced here prove the possibility of temporally independent tailor-made applications by means of a complete skin substitute for example in the area pharmascreening.