Native extracellular matrix (ECM) provides scaffolds for tissue engineering with natural architecture and biochemical composition. Maintaining the native ECM in decellularized tissues provides cues for cells, which promote their tissue specific arrangement and function. Several approaches have been used to decellularize ECM from the kidney in order to reestablish renal tissue but their comparability is hampered because methods for decellularization and assessment of ECM vary widely. Therefore, we applied a standardized immersion protocol to decellularize porcine kidney tissue with three detergents Triton X-100, SDS and sodium deoxycholate (SDC) at variable temperatures. For comparative analysis decellularization efficacies, structural preservation, composition and cell attachment and viability were analyzed. Structural ECM-conservation is strongly dependent on decellularization temperature, while preservation of glycosaminoglycans (GAG), collagens and cytokines was affected by the detergents used. GAG and collagens were best maintained by 1% SDS at 4 °C, whereas cytokines were best maintained in 1% SDC at 4 °C. Viability and attachment of human induced pluripotent stem cell derived renal precursor cells were best in SDC-ECM and thus not associated with the degree of GAG and collagen maintenance but the cytokine preservation. Based on structural and functional characteristics, we developed a scoring system that allows intra- and inter-study comparison of decellularization strategies. Application of the scoring system to our experimental data showed that decellularization with 1% SDS at 4 °C provided the highest structural and composition scores, while 1% SDC at 4 °C had lower structural and composition but a significantly better cell performance score. Inclusion of multiple published studies in the scoring matrix for comparison identified the highest structural and composition scores when decellularization was performed with SDS at low concentration, for a short period of time and at low temperature. Furthermore, the scoring system indicated that cell attachment and viability cannot be concluded from any other parameter and should therefore always be included in evaluation of decellularization strategies.

Advances in human pluripotent stem cell (hPSC) techniques have led them to become a widely used and powerful tool for a vast array of applications, including disease modeling, developmental studies, drug discovery and testing, and emerging cell-based therapies. hPSC workflows that require clonal expansion from single cells, such as CRISPR/Cas9-mediated genome editing, face major challenges in terms of efficiency, cost, and precision. Classical subcloning approaches depend on limiting dilution and manual colony picking, which are both time-consuming and labor-intensive, and lack a real proof of clonality. Here we describe the application of three different automated cell isolation and dispensing devices that can enhance the single-cell cloning process for hPSCs. In combination with optimized cell culture conditions, these devices offer an attractive alternative compared to manual methods. We explore various aspects of each device system and define protocols for their practical application. Following the workflow described here, single cell−derived hPSC sub-clones from each system maintain pluripotency and genetic stability. Furthermore, the workflows can be applied to uncover karyotypic mosaicism prevalent in bulk hPSC cultures. Our robust automated workflow facilitates high-throughput hPSC clonal selection and expansion, urgently needed in the operational pipelines of hPSC applications.

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