| 【Backgrounds】Trauma usually causes structural damage to tissues,triggering serial pathological events,including inflammation,blood supply disruption and cell loss.The healing or damage repair depends both on the nature of tissues and injury levels.For some special tissues,especially those with limited regenerative capacity,it is difficult for damage repair through self-repair ability,such as traumatic brain injury（TBI）.As is known,trauma on brain can cause a series of cascade reactions such as intracerebral hemorrhage,increased intracranial pressure,nerve excitatory toxicity and inflammation within minutes to hours.For the complicated pathological changes after TBI,it is crucial to develop a potent agent of wide protection against multiple neural damages.However,there is hardly a drug to meet the demand.Actually,an effective drug to inhibit brain injury or promote brain repair is still unavailable in clinic at present.Mesenchymal stem cells（MSCs）provide a potential strategy for protection and repair of those complicated tissue injuries,including TBI,due to their potent paracrine function.It has been confirmed that MSCs could secrete a variety of damage repair factors,such as angiogenic factor VEGF and b FGF,anti-inflammatory IL-10 and TSG-6,neuroprotective G-CSF,and so on.Therefore,both MSCs and derivates were extensively investigated for tissue repair in the past years.However,the paracrine function of conventional cultured MSCs in tissue culture dishes or plates was very restricted,and some cytokines are not even secreted.Activating the paracrine function to enhance the therapeutic potential of MSC is significant in the field of stem cell therapy,which has received extensive attention in recent years.Among various methods previously reported for MSC regulation,three-dimensional（3D）cultivation as spheroids should be the most effective one which was found both in clinical and laboratory studies.By present,the most classic method for scaffold-free 3D MSC spheroid formation was still the hanging drop culture.However,complex operation and low yield of spheroids formation of the method severely hinder its therapeutic application.Herein,inspired by the structure of sunflower disk,a microchamber-array plate（MCAP）was designed and fabricated through 3D printing.With the MCAP,large-scale production of homogenous MSC spheroids was achieved simply.In addition,conditioned medium（CM）which is abundant of therapeutic factors from MSC spheroids could be collected in large quantities through the MCAP.Multimodality of methods confirmed that MSC paracrine function was systematically and significantly enhanced,as a potent protection against multiple neural injuries in vitro and TBI in vivo was observed.Collectively,the study established a practical method to boost the therapeutic potential of MSC in large scale,which will be of great value for TBI and other tissue repair.【Objective】The study provided a practical and simple tool for large-scale production of enhanced MSCs,which is promising in stem cell therapy for various tissue injuries.【Method】1.The MCAP was designed with Rhino 6 and 3D printing was carried out using fused laminated FDM with ABS material.Subsequently,MCAP was coated with agarose and used for MSC 3D culture.The MCAP was improved based on the morphology,quantity and operation difficulty of the collected spheroids.MSC spheroids and conditioned medium were collected after 3 days of culture.The collected MSC spheroids were compared with the MSC spheroids cultured by HD,a traditional 3D culture method,in terms of particle size,morphology and viability.2.RNA was extracted from MSC of different densities in traditional 2D culture,HD culture and MCAP culture,and the expression levels of nutritional factors such as BDNF,VEGF,b FGF,SDF-1 and PLGF were detected by q PCR.RNA-seq was performed on MSCs under the optimal culture conditions of the three cultures to systematically compare the paracrine function of MSCs.The conditioned medium under this condition was detected by protein chip,and the spheroids under this condition were stained by immunofluorescence to compare the paracrine function of MSC under different culture methods from the gene and protein levels.3.CM was collected under the optimum culture conditions of traditional 2D,HD and MCAP culture methods.The protective effects of CM from different sources were compared in the injury model of mouse hippocampal neuron cell line HT22.The protective effect of CM was compared by observing cell morphology,cell viability and cell damage markers in complex injury model（stretch injury）.Apoptosis,necrosis and ferroptosis models were used to further compare their protective effects on specific injuries.The effect of CM on vascular remodeling after injury was compared by vascular venous endothelial cell migration and vascular formation models.4.MCAP-CM was collected for dialysis using 3KD dialysis bags,the by-products were removed and the nutrient factors were concentrated in different degrees.Mice were injected with different pretreated CM immediately after TBI.12h later,tissues from the injured site were collected,and markers of ferroptosis,apoptosis and inflammatory were detected by q PCR.The optimal treatment group was selected for RNA-seq to systematically detect the therapeutic effect of CM on TBI.5.Evans blue staining was performed 12h after injury to observe the effect of r CM on the recovery of blood-brain barrier function.Immunofluorescence staining（CD31,Iba1,Arg-1）was performed 7 days after injury to detect the effect of r CM on vascular remodeling and inflammation after TBI.Behavioral tests such as balance beam,rotating rod,elevated plus maze and new object recognition were performed at different time points within 1month after injury to detect the effects of r CM on motor,emotional and cognitive functions of TBI mice.【Result】1.Inspired by the structure of sunflower disk which could produce mass similar sunflower seeds,a MCAP was designed and fabricated through 3D printing.Different from conventional tissue culture dishes which have a flat bottom,the bottom of the MCAP was built with many uniformly arrayed microchambers,that nearly a thousand chambers were fabricated in a 125x85mm plate.The microchamber was improved for4 times.Finally,the microchamber was large at the top and small at the bottom.When the opening at the top was 3mm square,the middle of the chamber was inverted prism-shaped,the bottom of the chamber was a 2mm hemisphere,the depth of the microchamber was 1.5mm,and the adjacent ventricular septum was linear,the cells could be uniformly settled into the hemispheres at the bottom of the microchamber to form a large number of regular and uniform cell spheres.2.By hanging drop culture（HD）,a dish of 100mm-diameter（～78.5 cm2 in area）can produce～20 MSC spheroids,while a MCAP（～100 cm2 in area）can produce～1000 spheroids.The production of MSC spheroids was increased by 40～50 times.In morphology,the size of the MSC spheroids from MCAP was more uniform.The coefficient of variation of MSC spheroids size from HD was 0.1895,which was not in line with the normal distribution,while it was 0.0842 for that from MCAP,which was in line with the normal distribution,indicating that the MSC spheroids produced by MCAP were more homogeneous.After three days cultivation,Live/Dead staining showed that although cell viabilities were maintained for a high level for both HD and MCAP culture,cell viability was significantly higher in MCAP culture than that in HD.3.In HD culture,the optimal cell density was 6×104/m L,while it was 1.2×105/m L for MCAP.RNA-sequencing was employed to compare the consistency of MSC paracrine function between the two culture methods systematically,which showed that there was a high similarity between MSC spheroids from HD and MCAP culture（R>0.8）.Then,KEGG analysis was performed on the differently expressed genes on cells from HD,MCAP and conventional 2D culture.It was demonstrated that genes related to environmental information processing pathway was significantly enriched.KEGG analysis was further conducted and showed that pathways regulating cell proliferation,differentiation and survival,such as PI3K-Akt,c GMP-PKG and JAK-STAT,were significantly enriched.The results indicated that 3D culture was more conducive to stem cell development.In addition,the pro-angiogenesis genes were also significantly over expressed both in HD and MCAP cultured MSC spheroids.In the analysis of genes related to nervous system,both HD and MCAP cultures resulted in the significant enrichment of neuroactive gene set.4.The protein chip analysis obtained consistent results with RNA-sequencing that PI3K-AKT and angiogenesis pathways were significantly enriched.RT-PCR was further used to determine the expression of the representative cytokines and showed that most of them were upregulated by tens to hundreds of times,including neurotrophic factors BDNF,GDNF and PLGF,nerve regeneration-promoting factors G-CSF,SCF and SDF-1,angiogenesis factors b FGF,VEGF and HGF,anti-inflammatory factors IL-10 and TSG-6 and antioxidant factors IGF-1.Immunofluorescence staining also showed that the MCAP-cultured stem cell spheroids contained a large amount of cytokine proteins.The above studies indicated that MCAP exerted a potent regulation on MSC paracrine function,similar to classic HD culture method.5.CM from 2D,HD and MCAP were added to stretched cells and compared for their protective effects in the injury model.Mechanically damaged hippocampal neural cells（HT22）had reduced synapses and some cells were detached from the bottom of the dish,while HT22 cells protected by different CM remained in normal morphology.CCK8 assay showed that stretch significantly reduced viability of ht22.All of the differently derived CM demonstrated a protective effect on stretched cells,but MCAP-derived CM（AP-CM）demonstrated the most significant protection.The expression levels of proteins related to ferroptosis,apoptosis and necrosis were examined by western blot.These proteins were significantly highly expressed in the injury group,and the most significant downregulation of these proteins was observed in the AP-CM group among the treatment groups,indicating the best protective effect of AP-CM again.Flowcytometry analysis of apoptosis cells achieved consistent results with CCK8 assay.Three specific death models were then induced for further test,including ferroptosis,apoptosis and necrosis,all of which were key pathological events occurring after TBI.In Erastin-and RSL3-induced ferroptosis models,AP-CM exerted the best protection that the reduced cell viability by ferroptosis inducers was rescued to the greatest extent.In other cell death models,staurosporine-induced apoptosis and H2O2-induced necrosis,the best protective effect on cell viabilities were also observed in AP-CM treated group.The results suggested that AP-CM was even better than CM from HD culture in cell protection against multiple injuries.Then,the promotion effect on tissue repair was tested using angiogenesis model in vitro,because vascular reconstruction is a key event for injury repair.Cell scratch test was firstly performed to evaluate the effects of AP-CM on endothelial cell migration.Compared with 2D and HD culture-derived CM,AP-CM significantly promoted the migration of HUVEC.Next,endothelial tube formation experiment was performed.Tube formation assay showed that there were much more tube structures in AP-CM treated group than the other groups.The results indicated that MCAP cultured MSC was also better in promoting angiogenesis than 2D and HD-cultured ones.6.In order to refine the CM and remove adverse small molecules,the AP-CM was firstly centrifuged to remove cell debris,followed by dialysis with a 3k D dialysis bag and concentrated by sucrose.After dialysis,the color of CM changed from pink to nearly transparent,that is because phenol red was removed.For acute intervention of TBI,the refined CM（r CM）was intravenously injected after injury.12h after injection,some brains were dissected and injured tissues were collected for evaluation.RT-PCR showed that the original concentration of r CM after dialysis had the better therapeutic effect than concentrated ones.It could play protective roles such as anti-inflammatory and anti-necrosis.Thus,non-concentrated r CM was used in the following therapeutic experiments.7.RNA sequencing was performed on the injured tissues at 12h after surgery.GO analysis showed that negative regulation of neuron apoptotic process and negative regulation of neuron death pathways were significantly enriched in the r CM group.GSEA analysis showed that PDGF related to angiogenesis was enriched in the r CM group and NOS2 related to inflammation was enriched in the TBI group.Differential gene analysis showed that r CM-treatment upregulated anti-apoptosis,promoting nerve regeneration,vascular remodeling and anti-inflammation genes,while it inhibited pro-apoptosis and pro-inflammation genes.Meanwhile,Evans blue staining was performed to evaluate the integrity of brain-blood barrier（BBB）,which was closely associated with acute injury level of brain.Compared with non-treated TBI mice,the Evans blue stained area and the optical density value were smaller in r CM treated mice,indicating that r CM could promote the rapid recovery of BBB.Collectively,the results indicated that r CM exerted multiple protective effects on brain tissue at acute stage of TBI,consistent with the in vitro cell experiments.After1week of injury,the vascular reconstruction was evaluated on brain slice.The results showed that r CM could promote vascular remodeling at the injured site,which was beneficial for the repair of damaged tissues.8.To provide more evidence for the functional improvement of TBI mice,the brain tissues were further analyzed during the recovery after TBI.In comparison,smaller holes were observed at the injury site of r CM-treated mice at 1 week,indicating that r CM can reduce tissue loss and promote the tissue repair after TBI.Further,the activation of microglia was assessed by immunofluorescence staining with Iba1 and Arg-1.As is known,the subtypes of microglia were crucial for repair of injured brain tissue.It was shown that the ratio of M2 type microglia in r CM treated brains was significantly higher than that in non-treated ones.Co-localization of Ibal and Arg-1was frequently detected in r CM treated brains,while it was rarely detected in non-treated ones.The result indicated that r CM inhibited the overactivation of microglia and promoted their transformation to anti-inflammatory M2 type,which was also beneficial for brain repair.【Conclusion】1.We designed and fabricated a microchamber-array plate（MCAP）,which can simply achieve large-scale production of homogenous MSC spheroids,increasing the culture efficiency by～50 times compared with classic hanging drop culture;2.MCAP systematically enhanced the paracrine function of MSCs from gene to protein levels;3.In vitro,MCAP cultured MSCs demonstrated a potent protection against multiple injuries of neural cells;4.In TBI mice,MCAP cultured MSCs demonstrated great therapeutic benefits for tissue repair;5.The study provided a practical and simple tool to boost the therapeutic potential of MSCs,which would meet the therapeutic demand in scale and is promising in clinical t application. |
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