Separation And Transdifferentiation Of Bone Marrow Stromal Cells In Microfluidic Chip And Their Efficacy On Neurite Outgrowth

Peripheral nerve injuries are an economic burden for society in general andfunctional recovery following microsurgical reconstruction is unsatisfactory. Morerecently, promising repair strategy have been achieved using tissue engineeredperipheral nerve (TEPN) for the treatment of peripheral nerve injury.Treatment by the implantation of Schwann cells (SCs), neurotrophic factors andbiological materials is used to induce nerve regeneration at nerve defects nowadays.SCs provide structural support and guidance for peripheral nerve regeneration byreleasing neurotrophic factors, but the limited Schwann cell resource and the xenogenicgraft immunorejection reaction hindered its further application. Bone marrow stromalcells (BMSCs) are an attractive alternative candidate because they exhibit severalimportant and potential advantageous features both in PNS and CNS regeneration. Theycan be obtained easily, and display an unorthodox plasticity.There are several difficulties to be overcomed on BMSCs-based therapy forperipheral nerve regeneration. BMSCs are still couldn’t be effectively extracted andpurificated. The optimal induce factors combination for BMSCs differentiating intoSC-like cells is still uncertain. Microfluidic technology has proven very useful for highthroughput analysis at cellular level and was widely applied on cell-based research.On-chip-based microfluidic devices could be used for cell separation, microscalecultures, creating specific cellular microenvironments as well as analysis andmanipulation of biological samples.We planed to base on the microfluidic chip technology to solve these problems.(1)Develop novel microfluidic devices to optimize the operations of stem cells separationand purification.(2) Find out the best induce factors combination for BMSCs differentiating into SC-like cells.(3) Assess the functional performance of the optimalinduced SC-like cells on neurite outgrowth.The first part: BMSCs separation and purification on microfluidic chip.Objective: We designed and fabricated a novel electromagnetic separationmicrofluidic chip to efficiently remove magnetically tagged cells from suspension.Inorder to separate the BMSCs fastly and with a high purification, we firstly tested thecell sorting efficiency of this microfluidic chip.Methods: The design of the electromagnetic separation microfluidic chip wasinspired by the operation of fluorescence activated cell sorting (FACS) and magneticactivated cell sorting (MACS). To evaluate the capability of this chip, we performed anegative enrichment of tumor cells (SK-BR-3, breast carcinoma) suspended in anabundant population of leukocytes. Tumor cells and leukocytes were firstly stained withfluorescent antibody separately, and the samples were prepared by mixing a knownamount of leukocytes and tumor cells. The two cell populations were then mixed andincubated with CD45magnetic beads for specific labeling of leukocytes. Forquantitative analysis of cell separation by the electromagnetic separation microfluidicchip, we performed flow cytometry on samples before and after sorting. we performedflow cytometry on samples before and after sorting for quantitative analyses theefficiency of cell separation by the electromagnetic separation microfluidic chip. Wefinally obtained the optimal speed for cell sorting.Bone marrow mononuclear cells, which were just isolated from rat femurs, wereimmediately pumped through the electromagnetic separation microfluidic chip forenrichment of BMSCs. A part of the obtained CD271immunopositive and CD45immunonegative cells were charactered by flow cytometry for purity determination. Theother cells were operated for characterization of multi-directional differentiation.Results: The optimal cell separation rate we obtained by the electromagneticseparation microfluidic chip was the flow rate at4.0mL/h, where the sorting rate was(92.54±3.33)％. FACS analysis for BMSCs surface markers demonstrated that the cellswere positive for CD29(99.17%) and CD90(98.95%), but negative for CD45(0.76%).These screened BMSCs could differentiate into chondrocytes, osteocytes andadipocytes.Conclusions:1. The electromagnetic separation microfluidic chip can be operatedfor high throughput cell separation. The optimal cell input velocity is at4.0mL/h,where the sorting rate is (92.54±3.33)％.2. Primary BMSCs can be obtained with high purity through this microfluidic chip.The second part: BMSCs differentiating into SC-like cells on microfluidicchipObjective: Inorder to obtained the optimal compatibility concentration of theinduce factors for BMSCs differentiating into SC-like cells, we designed aconcentration gradient generation microfluidic chip for high throughput screening thebest combination of the induce factors.Methods: The microfluidic device was mainly composed of an upstreamconcentration gradient generator (CGG) and a downstream cell culture module. Thecombinations of the critical induce factors forskolin (FSK) and β-heregulin (HRG) wereat the different concentrations in downstream cell chambers. Rat BMSCs were culturedin the cell chambers for11days and then immunostaining with Schwann cell markersS100β and p75NTRseparately. The chamber, reflecting the individual concentration ofinduce factors, which showed the most immunopositive cells and expressed themaximal gray scale of S100β and p75NTRwas recognized with the optimal combination.Results: After the administration of induce factors, BMSCs changed from amonolayer of large, flat cells into a bipolar, spindle-like shape. Immunocytochemistryfor S100βand p75NTRimmunopositive cells on-chip demonstrated that the cell chambers,with the induce factors concentration of4.00μM FSK and250.00ng/ml HRG,indicated the largest number of S100β and p75NTRimmunopositive cells. And thenormalized fluorescent intensity per immunopositive cell was at the maximal gray scalein the same chamber.Conclusions:1. We built a concentration gradient generation microfluidic chip,and successful induced BMSCs into Schwann-like cells on-chip.2. We originallyobtained the optimal combination of the inducers concentration for BMSCsdifferentiating into SC-like cells based on the microfluidic device and meanwhileavoiding a large number of repeated tests.The third part: The functional performance of the optimal induced SC-likecells on neurite outgrowth.Objective: We built the DRG co-culture system to assess the neurite outgrowthperformance of the optimal induced SC-like cells, and used RT-PCR and ELISAmethodology to analyze relative neurotrophins secretion.Methods: DRG co-culture was established to detect the neurite outgrowthproperties of undifferentiated BMSCs (group A), SC-like cells cultured in conventional differentiate media (group B), SC-like cells cultured in optimal differentiate media(group C) and normal Schwann cells (group D). After24h co-culture, DRG neuriteswere immunostained with anti-βⅢ tubulin antibody, and the neurite outgrowth wasassessed using three independent parameters: percentage of process-bearing neurons,length of longest neurite and total neurite density. The mRNA levels of SCs markers(S100β and p75NTR) and neurotrophic factors (BDNF, NGF and NT-3) among the cellsin different groups were tested by RT-PCR. The conditioned media of105cells in groupA, B, C and D analyzed by ELISA using rat BDNF, NGF or NT-3sandwich ELISAkits.Results: Immunocytochemistry for βⅢ-tubulin showed a significant increase inthe neurite sprouting, length of the longest neurite and neurite density when DRGneurons were co-cultured with cells of group C as compared with control basal media (p<0.01) and cells of group A (p <0.01). Cells of group C promoted the more neuriteoutgrowth similar to normal Schwann cells (group D). RT-PCR results indicated thatthe levels of each expressed mRNA in the optimal induced SC-like cells (group C) werewere significantly higher than that in group A and B, and near to normal Schwann cells(group D). ELISA quantification indicated that BDNF, NGF and NT-3secreted by cellsin group C were at significantly higher levels than that in group A and B, and the levelswere similar to group D.Conclusions:1. The functional performance of the optimal induced SC-like cellson neurite outgrowth is similar to normal Schwann cells.2. This elevated functionalperformance may attribute to the up-regulated mRNA expression and secretion ofneurotrophins, such as BDNF, NGF and NT-3.