Three-dimensional cell culture and tissue engineering in a T-CUP (tissue culture under perfusion)

The aim of this study was to develop and validate a simple and compact bioreactor system for perfusion cell seeding and culture through 3-dimensional porous scaffolds. The developed Tissue Culture Under Perfusion (T-CUP) bioreactor is based on the concept of controlled and confined alternating motion of scaffolds through a cell suspension or culture medium, as opposed to pumping of the fluid through the scaffolds. Via the T-CUP, articular chondrocytes and bone marrow stromal cells could be seeded into porous scaffolds of different compositions and architectures (chronOS, Hyaff-11, and Polyactive) at high efficiency (greater than 75%), uniformity (cells were well distributed throughout the scaffold pores), and viability (greater than 97%). Culture of articular chondrocytes seeded into 4-mm thick Polyactive scaffolds for 2 weeks in the T-CUP resulted in uniform deposition of cartilaginous matrix. Cultivation of freshly isolated human bone marrow nucleated cells seeded into ENGipore ceramic scaffolds for 19 days in the T-CUP resulted in stromal cell-populated constructs capable of inducing ectopic bone formation in nude mice. The T-CUP bioreactor represents an innovative approach to simple, efficient, and reliable 3D cell culture, and could be used either as a model to investigate mechanisms of tissue development or as a graft manufacturing system in the context of regenerative medicine

HLA-DPA1*02:01~B1*01:01 is a risk haplotype for primary sclerosing cholangitis mediating activation of NKp44+ NK cells

Objective Primary sclerosing cholangitis (PSC) is characterised by bile duct strictures and progressive liver disease, eventually requiring liver transplantation. Although the pathogenesis of PSC remains incompletely understood, strong associations with HLA-class II haplotypes have been described. As specific HLA-DP molecules can bind the activating NK-cell receptor NKp44, we investigated the role of HLA-DP/NKp44-interactions in PSC. Design Liver tissue, intrahepatic and peripheral blood lymphocytes of individuals with PSC and control individuals were characterised using flow cytometry, immunohistochemical and immunofluorescence analyses. HLA-DPA1 and HLA-DPB1 imputation and association analyses were performed in 3408 individuals with PSC and 34 213 controls. NK cell activation on NKp44/HLA-DP interactions was assessed in vitro using plate-bound HLA-DP molecules and HLA-DPB wildtype versus knock-out human cholangiocyte organoids. Results NKp44+NK cells were enriched in livers, and intrahepatic bile ducts of individuals with PSC showed higher expression of HLA-DP. HLA-DP haplotype analysis revealed a highly elevated PSC risk for HLA-DPA1*02:01~B1*01:01 (OR 1.99, p=6.7×10-50). Primary NKp44+NK cells exhibited significantly higher degranulation in response to plate-bound HLA-DPA1*02:01-DPB1*01:01 compared with control HLA-DP molecules, which were inhibited by anti-NKp44-blocking. Human cholangiocyte organoids expressing HLA-DPA1*02:01-DPB1*01:01 after IFN-γ-exposure demonstrated significantly increased binding to NKp44-Fc constructs compared with unstimulated controls. Importantly, HLA-DPA1*02:01-DPB1*01:01-expressing organoids increased degranulation of NKp44+NK cells compared with HLA-DPB1-KO organoids. Conclusion Our studies identify a novel PSC risk haplotype HLA-DP A1*02:01~DPB1*01:01 and provide clinical and functional data implicating NKp44+NK cells that recognise HLA-DPA1*02:01-DPB1*01:01 expressed on cholangiocytes in PSC pathogenesis