Autofluorescence-based label-free cell counting method in suspension culture with microcarriers

Abstract To advance the industrialization of cultured meat and regenerative medicine, scalable and efficient cell culture techniques are essential. Among these, the suspension culture method using microcarriers has emerged as a promising approach for the large-scale cell culture technique. However, monitoring cell growth on the microcarriers remains challenging, particularly in developing cell counting techniques that can be seamlessly integrated into bioprocess workflows without cell detachment, fluorescence labeling and any parameter tuning in the analysis algorithm. In this study, we proposed a versatile image analysis-based cell counting method by using cellular autofluorescence without any parameter tuning. The proposed method estimates the number of cells by applying spatiotemporal averaging to the autofluorescence signals in the microscopic images. Using numerical and cell culture experiments, we demonstrated that the proposed method can estimates the number of cells accurately. This technique, which harnesses the ubiquitous autofluorescence inherent in living cells, offers a cost-effective and practical solution applicable to a broad range of fields requiring high-throughput cell quantification.