03865nas a2200709   4500000000100000000000100001008004100002260000900043653003200052653001200084653002800096653003500124653001600159653001800175653003000193653001100223653005100234653004200285653003000327653003300357653001700390653002800407653002000435100001900455700001800474700001800492700001800510700002000528700001700548700002800565700002300593700001400616700002200630700002700652700001600679700001400695700002600709700002000735700002000755700003100775700001900806700002000825700002400845700002600869700001900895700002000914700001500934700002200949700001800971700001700989700002001006700001901026700002001045700001901065700002001084700002001104245011701124300001101241490000701252520188201259022001403141       2025                            d  c202510aAnimal Testing Alternatives10aAnimals10aArtificial intelligence10aBiological Science Disciplines10aBioprinting10aDigital Twins10aHigh-throughput screening10aHumans10aLive-cell, high-content and real-time analysis10aMicrogravity and radiation simulators10amicrophysiological system10aPrimary cells and stem cells10aSpace Flight10aSpheroids and organoids10asystems biology1 aMathieu Vinken1 aDaniela Grimm1 aSarah Baatout1 aBjorn Baselet1 aAfshin Beheshti1 aMarkus Braun1 aAnna Catharina Carstens1 aJames A. Casaletto1 aBen Cools1 aSylvain V. Costes1 aPhoebe De Meulemeester1 aBartu Doruk1 aSara Eyal1 aMiguel J. S. Ferreira1 aSilvana Miranda1 aChristiane Hahn1 aSinem Helvacıoğlu Akyüz1 aStefan Herbert1 aDmitriy Krepkiy1 aYannick Lichterfeld1 aChristian Liemersdorf1 aMarcus Krüger1 aShannon Marchal1 aJette Ritz1 aTheresa Schmakeit1 aHilde Stenuit1 aKevin Tabury1 aTorsten Trittel1 aMarkus Wehland1 aYu Shrike Zhang1 aKarson S. Putt1 aZhong-Yin Zhang1 aDanilo A. Tagle00aTaking the 3Rs to a higher level: replacement and reduction of animal testing in life sciences in space research  a1085740 v813 aHuman settlements on the Moon, crewed missions to Mars and space tourism will become a reality in the next few decades. Human presence in space, especially for extended periods of time, will therefore steeply increase. However, despite more than 60 years of spaceflight, the mechanisms underlying the effects of the space environment on human physiology are still not fully understood. Animals, ranging in complexity from flies to monkeys, have played a pioneering role in understanding the (patho)physiological outcome of critical environmental factors in space, in particular altered gravity and cosmic radiation. The use of animals in biomedical research is increasingly being criticized because of ethical reasons and limited human relevance. Driven by the 3Rs concept, calling for replacement, reduction and refinement of animal experimentation, major efforts have been focused in the past decades on the development of alternative methods that fully bypass animal testing or so-called new approach methodologies. These new approach methodologies range from simple monolayer cultures of individual primary or stem cells all up to bioprinted 3D organoids and microfluidic chips that recapitulate the complex cellular architecture of organs. Other approaches applied in life sciences in space research contribute to the reduction of animal experimentation. These include methods to mimic space conditions on Earth, such as microgravity and radiation simulators, as well as tools to support the processing, analysis or application of testing results obtained in life sciences in space research, including systems biology, live-cell, high-content and real-time analysis, high-throughput analysis, artificial intelligence and digital twins. The present paper provides an in-depth overview of such methods to replace or reduce animal testing in life sciences in space research.  a1873-1899