Establishment And Optimization Of An In Vitro Inflammatory Model Of A Novel Human Immortalized Microglia HM-SV40

China has the largest population of cerebrovascular disease patients,and the highest incidence rate of stroke in the world.As the aging of the Chinese population is gaining speed,the increase of other neurodegenerative diseases(eg.Alzheimer's disease,Parkinson's disease)is also quite alarming.Among the many physiopathological charateristics of both acute and chronic neuronal injury,over-activation and prolonged activation of the inflammatory response is one of the primary features.CNS inflammation is implicated in almost all neurovascular and neurodegenerative diseases.Owing to the presence of the blood-brain barrier(BBB),inflammatory cells of the peripheral innate immune system are not able to enter the central nervous system(under normal circumstances).As a result,glial cells,microglia in particular,constitute the major players of the CNS inflammatory response.Immortalized murine microglial cell lines(e.g.: BV2)used to be important tools for studying CNS inflammatory response in vitro.However,there is a growing body of evidence that murine microglial cell,especially the immortalized ones,behave distinctively from their human counterpart,such as different gene expression spectra,different intensity and duration of inflammatory response,different composition of secreted inflammatory cytokines/chemokines.Cell lines like BV2 are also poor model systems for predicting in vivo efficacy of drug candidates designed for suppressing CNS inflammation.On the other hand,human primary microglia is extremely difficult to obtain and the derivation of human induced pluripotent stem cell(iPSC)into microglia is very expensive.Thus,murine primary microglia and human immortalized microglial cell line become the cost-efficient choices.However,human immortalized microglial cell lines are still insufficiently studied,and the culturing and operating protocols of murine primary microglia have not been updated for more than a decade.The primary objectives of the studies in this thesis are: 1)establish and optimize the culture and operation protocol of human immortalized microglia cell line(HM-SV40)from Abm Goods company(Canada);2)Optimize current operation protocols of murine primary microglia and evaluate the applicability of various newly emerged culturing material and bioanalytical approaches in the study of murine primary glial cultures.Part ?: We established and optimized the culture and operation protocol of human immortalized microglia cell line(HM-SV40).We first validated the expression of microglia-specific biomarker TREM2 with qPCR.The qPCR assays also showed that this cell line carries the canonical TLR family receptors,and LDLR family receptors.Interestingly,conventional inflammatory stimulants,such as lipopolysaccharide(LPS),PolyI:C and phytohemagglutinin(PHA),were not able to induce discernable inflammatory response in HM-SV40 cells,as shown by both qPCR and ELISA assays.After a systematic search,we discovered that conditioned media from activated murine microglia or macrophage could induce strong inflammatory response from HM-SV40 cells.With the help of multiplex cytokine assays,we eventually found that TNF-alpha and IFN-gamma were able to efficiently activate HM-SV40 cells,which were verified by transcriptomic RNA-seq analysis.Results from this part of thesis are vitally important for future study of HM-SV40 cell line in inflammatory response.Part ?: We obtained murine microglial cells with both conventional mechanical approach and the newly developed MACS approach.These cells were responsive to LPS stimulation and the inflammatory response could be suppressed by apoE-mimetic peptide COG1410.We then evaluated the effect of a series of new culturing materials,such as Matrigel,Collagen I,on the culture of primary murine microglial cells.We also developed special protocol for improving the inter-batch stability of these cell cultures.Our results from multiplex cytokine assay and qPCR assay showed that Matrigel is superior to other conventional coating materials(e.g.: Poly-D-Lysine)for its ability to better mimic extracellular matrix environment.We also showed substantial synergistic effect between astrocyte and microglia.Our results from this part of thesis provides a new set of protocol for future study of murine primary glial cultures.Part ?: We conducted preliminary study of apoE-mimetic peptide CN-105 on the most widely used microglial cell line – BV2.Our results showed that CN-105 could not suppress inflammatory response in BV2.Part ?: We used surface plasmon resonance(SPR)approach to determine the binding affinity between LDLR-related protein 1(LRP1)'s ligand bind modules,and various apoE and apoE-mimetic peptides.The results showed that ApoE and apoE-mimetic peptide COG1410 could bind LRP1 with moderate affinity,while we did not observe any sign of binding for CN-105.In conclusion,the studies in this thesis focused primarily on the optimization of culturing and operating techniques of human and murine microglial cell models,with emphasis on inflammatory response.Our results provided a set of more efficient and reproducible protocols for future in vitro studies of CNS inflammation.