Development Of A Biomimetic Microenvironment Co-Culture System To Enhance The Hair Inductivity Of Dermal Papilla Cells

BackgroundThe complex 3D microenvironmental conditions to which dermal papilla cells(DPCs)are exposed in vivo not only include 3D cell aggregation and cell-extracellular matrix interactions,but also the cytokine-mediated crosstalk between DPCs and neighbouring cells(i.e.,hair matrix cells(HMCs)and dermal sheath cup cells(DSCCs)).Unfortunately,the research on the molecular mechanisms that the engineered 3D microenvironment orchestrates DPCs fate has been proceeded slowly due to lack of suitable cell culture system and study platform.Moreover,the use of hair follicles(HFs)engineered from DPCs for progressive hair loss treatment is also limited by the lack of efficient and general large-scale preparation strategy.Materials and methods1.Construction and evaluation of Matrigel three-dimensional culture model of DPCsP4 DPCs were mixed gently with original Matrigel and then spotted the mixture as a droplet on the membranes’ lower surface for three-dimensional culture.Dermal papilla cell aggregations(DPAs)can be obtained through culture.Then the DPAs morphology and behavior were continuously observed,and the optimal concentration were selected from four groups at variable concentrations.The activity of DPAs,the expression of essential marker genes and hypoxia-dependent changes were detected to evaluate the performance of the model(Model group).Two-dimensional culture(Control group)and hanging drop method(HDrop group)were adopted as controls.2.Construction of the 3-D co-culture system mimicking DPCs microenvironment and large-scale preparationThe inserts of Model cultured one day were transferred into the wells which DSCCs were adherent one day.HMCs were then layered on the upper chamber and tri-cultured for an additional 6 days.The DPAs were manufactured by using the microneedle injection instrument with multiple needles.The key genes expression of DPAs and the HFs inductivity in vivo were detected.Two-dimensional culture(Control group),Matrigel model(Model group),and primary DPCs(P0 group)were compared.3.Explore the influence of biomimetic microenvironment on the biological functions of DPCsAfter seven days culture,collect different DPCs/DPAs(P4,Model,System,P0 group)for high-throughput sequencing analysis.Results1.A Matrigel three-dimensional culture model of DPCs was successfully constructedIn Matrigel,DPCs spontaneously aggregated into cell aggregates of various sizes(50-200μm).The optimal formation conditions of DPAs were determined to be 1×104 cells/μL at D7.DPAs in the model group maintained good cell viability and exhibited higher proliferation,fewer apoptosis/hypoxia-related factors.More importantly,the expression of key genes of DPCs is not only significantly higher than the two-dimensional cell culture method,abut also better than hanging drop method(P<0.05).2.The three-dimensional co-culture system mimicking DPCs microenvironment and the large-scale preparation were successfully accomplishedSurprisingly,compared with the Model group,some specific essential genes(ALP,NCAM,SOX2,and β-catenin)in the System group were further upregulated to varying degrees(P<0.05).Large-scale production of DPAs was achieved by introducing a micro-needle automatic injection device with multiple needles for the first time.After co-cultivation with DSCCs and HMCs,a large number of DPAs with high inducibility were formed.In the patch assay,the regeneration and maturity of HFs of the biomimetic system was second only to the primary DPCs group,which was significantly better than the model group.3.The influence of biomimetic microenvironment on the biological functions of DPCs was successfully generalizedThe mRNA expression profiles of DPCs/DPAs were harvested by using Illumina high-throughput sequencing.The related terms of Wnt signaling pathway and BMP pathway were respectively enriched in the differential genes that were gradually up-regulated/down-regulated in the Control-Model-System-P0 sequence.The biomimetic microenvironment(System VS Control)could restore the hair inducing ability of DPCs by simultaneously activating the classic Wnt pathway and inhibiting the BMP pathway.ConclusionsCompared with monolayer culture and hanging drop method,Matrigel model simulates the aggregation state of cells,and significantly enhances the DPCs biological characteristics.By activating Wnt signaling and inhibiting the BMP signaling pathway,the biomimetic microenvironment significantly improves the hair-inducing potential of 2D-expanded DPCs.This system realizes the large-scale preparation of DPCs with good biological functions and provides a new solution for the treatment of large-area hair loss.