Umbilical cord is an abundant source of perinatal, plastic adherent mesenchymal stem cells (UC-MSCs). UC-MSCs exhibit robust stemness and strong immunosuppressive and regenerative effects in vivo. This protocol describes enzymatic and mechanical dissociation of umbilical cord matrix (Wharton's jelly) that results in efficient isolation of large numbers of fresh nucleated umbilical cord regenerative cells (UC-RCs) that, when cultured on plastic, exhibit similar characteristics of UC-MSCs. This protocol potentially alleviates the need for culture expansion to obtain large numbers of cells required for clinical application. Dissociation is achieved with a blend of collagenase and neutral proteases with agitation at 37 °C in a semi-automatic system. Average expected yield is 1.65 × 10(6) cells/g tissue with 93 % viability. This protocol has been successfully used to isolate an uncultured nucleated regenerative cell population (also referred to as stromal vascular fraction or SVF) from surgically debrided skin and from human, equine, and canine adipose tissue. The procedure requires less than 30 min for tissue dissection and less than 100 min for cell extraction. Quickly obtaining a large number of UC-RCs that have pluripotent differentiation capacity without the complexity and risks of culture expansion could simplify and expand the use of UC-RCs in clinical as well as research applications.