BackgroundThe study of the distinct structure and function of the human central nervous system, both in healthy and diseased states, is becoming increasingly significant in the field of neuroscience. Typically, cortical and subcortical tissue is discarded during surgeries for tumors and epilepsy. Yet, there is a strong encouragement to utilize this tissue for clinical and basic research in humans. Here, we describe the technical aspects of the microdissection and immediate handling of viable human cortical access tissue for basic and clinical research, highlighting the measures needed to be taken in the operating room to ensure standardized procedures and optimal experimental results.MethodsIn multiple rounds of experiments (n= 36), we developed and refined surgical principles for the removal of cortical access tissue. The specimens were immediately immersed in cold carbogenated N-methyl-D-glucamine-based artificial cerebrospinal fluid for electrophysiology and electron microscopy experiments or specialized hibernation medium for organotypic slice cultures.ResultsThe surgical principles of brain tissue microdissection were (1) rapid preparation (<1 min), (2) maintenance of the cortical axis, (3) minimization of mechanical trauma to sample, (4) use of pointed scalpel blade, (5) avoidance of cauterization and blunt preparation, (6) constant irrigation, and (7) retrieval of the sample without the use of forceps or suction. After a single round of introduction to these principles, multiple surgeons adopted the technique for samples with a minimal dimension of 5 mm spanning all cortical layers and subcortical white matter. Small samples (5–7 mm) were ideal for acute slice preparation and electrophysiology. No adverse events from sample resection were observed.ConclusionThe microdissection technique of human cortical access tissue is safe and easily adoptable into the routine of neurosurgical procedures. The standardized and reliable surgical extraction of human brain tissue lays the foundation for human-to-human translational research on human brain tissue.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00701-023-05611-9.