Classical intracellular delivery methods such as transfection and transduction are inefficient, particularly with confluent cells and organoids, and lack cell type‐specific programmability. We demonstrate that an innovative methodology called calcium shock (CaSh) dramatically improves particle delivery into single cells, colonies, and organoids, and enables programmable delivery (CaSh‐Pro) into specific cell types within heterocellular populations. Calcium shock works by increasing endocytotic uptake while simultaneously disarming cell‐cell junctions. CaSh‐Pro further incorporates specific molecular targeting agents and amphiphilic peptides for preferential editing of different cell types. Calcium shock improves expression of plasmid, ribonucleoprotein, or adeno‐associated viral vectors with minimal toxicity in intact organoids representing diverse lineages. CaSh and CaSh‐Pro provide simple, versatile protocols for genome editing in complex systems, to enable biological discovery and therapeutic development. Classical transfection and transduction are inefficient, particularly with confluent cells and organoids, and lack cell type‐specific programmability. This study presents calcium shock (CaSh), a method that dramatically improves particle delivery into single cells, colonies, and organoids. CaSh is further utilized to enable programmable delivery (CaSh‐Pro) into specific cell types within heterocellular populations.

Introduction: Extensive thermal injury remains a formidable clinical challenge, primarily due to the profound deficit of autologous donor skin, which necessitates prolonged hospitalization and escalates healthcare expenditures. While human embryonic stem cells (hESCs) offer a theoretically inexhaustible source for regenerative therapy, optimizing their differentiation and engraftment remains critical for clinical translation. Methods: We used a three-stage protocol to induce the differentiation of hESCs into keratinocytes (KCs). To optimize the delivery of hESC-derived keratinocytes (EKCs), human dermal fibroblasts (HFBs) were utilized to provide essential extracellular matrix (ECM) and microenvironmental support. The two cell types could self-assemble into 3D spheroids. After optimizing the size and cell proportion, these spheroids were subsequently transplanted onto full-thickness dorsal wounds in immunodeficient mice to evaluate their regenerative capacity. Results: hESC-derived keratinocytes exhibited the expression of stage-specific epidermal markers, confirming high differentiation efficiency. In vitro, EKCs demonstrate the capacity to form stratified epidermal structures. By self-assembling into spheres with dermal fibroblasts, the EKCs demonstrated successful engraftment and sustained survival in vivo. The transplantation of these 3D spheroids significantly accelerated wound closure and re-epithelialization compared with controls. Conclusions: This study establishes a robust cell therapy approach characterized by a short preparation cycle with high differentiation efficiency and high transplantation survival rate, offering a novel strategy for the treatment of extensive skin defects.

Background: The pathology of Alzheimer’s Disease (AD) is characterized by aggregates of amyloid beta (Aβ) peptides and neurofibrillary tau tangles. Increased blood-brain barrier (BBB) permeability and reduced Aβ clearance, which signal neurovascular dysfunction, have also been proposed as early markers of AD. Despite intense scrutiny, the mechanisms of AD remain elusive and novel treatments that address core symptoms of dementia are limited. New alternative methods (NAMs) aim to develop in-vitro translational models that recapitulate human pathology more accurately than previous models and could contribute to the development of new therapies. Methods: Here, we developed a NAM model of the cortical neurovascular unit (NVU) using brain cells derived from human induced pluripotent stem cells (hiPSCs) from a patient with AD and a healthy individual. Differentiated neurons, astrocytes, pericytes, microglia, and brain-like microvascular endothelial cells were cultured in a microphysiological system to create a brain-chip model to evaluate NVU-related endpoints. Results: Compared to control, AD brain-chips had reduced claudin-5 and ZO-1 expression and increased paracellular permeability. AD brain-chips also had decreased activity of the efflux transporter P-glycoprotein (P-gp), but its expression was unchanged. In AD brain-chips, levels of Aβ42, total tau, and p-tau 181 were decreased in protein lysates from the brain channel, while levels of total tau and p-tau 181 were increased in protein lysates from the vascular channel. Finally, AD brain-chips had increased levels of the proinflammatory markers IL-6 and MCP-1 in effluent from both brain and vascular channels. Conclusion: In this brain-chip model, we showed Aβ-independent NVU dysfunction that was related to neuroinflammation and vascular tau accumulation. This study demonstrates the utility of the brain-chip model to evaluate changes in NVU functions induced by AD-like pathology and highlights donor-specific responses associated with the use of hiPSC-derived models.