Ex vivo expansion of CD34^+ cells and immunocytes from umbilical cord blood

Objective： Umbilical cord blood stem cell transplantation （CBSCT） has approached significant success in leukemia treatment, but it is associated with higher rates of delayed or failed engraftment and relapse. This may be caused by immature immune calls of umbilical cord blood. We try to expand stem/progenitor calls and T, NK, DC immunocytes from umbilical cord blood for transplantation and immunotherapy. Methods： CB MNCs were cultured and analyzed for progenitor/stem calls, immunocytes at day 0, 3, 7 and 14 by using flowcytometry. Results： The combinations of SCF, IL-3, IL-6, plus IL-2 or/and IL-4 showed significantly expanded results both for UCB MNCs and CD34^＋ cells. CD34^＋ percentage went up from fresh CB 1.6% to the highest group E （SCF＋IL-3, 6, 2, 4） 11.1%. The average expansion multiples of CD34^＋ calls at 7th culture days were from 10 to 50 （SCF＋IL-3, 6, 2, 4）. The CD3^＋ T cells was （18.7±4.3）% in fresh cord blood, and decreased sharply in the medium without cytokine, while markedly increased in groups with cytokines combination, in group B, E, G and F, their level were about 2 times of fresh control. The fresh UCB contained （3.6±1.9）% CD56^＋ calls, NK cells only were expanded in groups with IL-2. DCs markers CDla, CD80, CD83 and CD86 expressed a lower level at day 3 in all test groups, and then increased sharply in groups E, F and G with IL-4 cytokin at 7th culture days. Conclusion： T calls, NK cells and DCs as well as stem/progenitor calls could be expanded in the same medium from CB MNCs with the combinations of cytokines. The combination of SCF, IL-2, IL-3, IL-6 and IL-4 showed a balancaable expansion result of both CD34^＋ cells and immunocytes at 7th culture days.

Isolation and <i>Ex Vivo</i>Expansion of Human Hematopoietic Stem Cells Derived from Umbilical Cord Blood

Umbilical cord blood (UCB) is a current major source of hematopoietic stem cells (HSCs) for cell transplantation therapy. Cell transplantation with HSCs derived from UCB is advantageous over transplantation with HSCs from adult tissues. However, the low number of HSC derived from a single unit of UCB limits its application. Thus, ex vivo expansion is a good option to create more UCB HSCs for clinical application. The strategies for HSC expansion in vitro focus on mimicking the composition and structure of HSC natural niche by enhancing self-renewal and inhibiting lineage differentiation of HSCs. In the past decade, the mechanisms of the interaction between HSC and the natural niche have been deeply investigated. This great progress in basic research has led to advancements in UCB HSC ex vivo expansion. In addition, the biological characteristics of the originally isolated UCB HSCs correlate with outcome of subsequent ex vivo expansion. In this paper, we summarize the late progress achieved in isolation and ex vivo expansion of UCB HSCs. Importantly, we attempt to provide an impact and practicable procedure to expand UCB HSC in vitro from isolation of original HSCs to identification of expanded HSCs.

Ex vivo expansion of hematopoietic stem and progenitor cells: Recent advances

Hematopoietic stem cells(HSCs) have become the most extensively studied stem cells and HSC-based cellular therapy is promising for hematopoietic cancers and hereditary blood disorders. Successful treatment of patients with HSC cells depends on sufficient number of highly purified HSCs and progenitor cells. However, stem cells are a very rare population no matter where they come from. Thus, ex vivo amplification of these HSCs is essential. The heavy demands from more and more patients for HSCs also require industrial-scale expansion of HSCs with lower production cost and higher efficiency. Two main ways to reach that goal:(1) to find clinically applicable, simple and efficient methods(or reagents) to enrich HSCs;(2) to find new developmental regulators and chemical compounds in order to replace the currently used cytokine cocktails for HSCsamplification. In this Editorial review, we would like to introduce the current status of ex vivo expansion of HSCs, particularly focusing on enrichment and culture supplements.

Ex vivo liver resection and auto-transplantation as an alternative for the treatment of liver malignancies: Progress and challenges

Hepatectomy is still the major curative treatment for patients with liver malignancies.However,it is still a big challenge to remove the tumors in the central posterior area,especially if their location involves the retrohepatic inferior vena cava and hepatic veins.Ex vivo liver resection and auto-transplantation(ELRA),a hybrid technique of the traditional liver resection and transplantation,has brought new hope to these patients and therefore becomes a valid alternative to liver transplantation.Due to its technical difficulty,ELRA is still concentrated in a few hepatobiliary centers that have experienced surgeons in both liver resection and liver transplantation.The efficacy and safety of this technique has already been demonstrated in the treatment of benign liver diseases,especially in the advanced alveolar echinococcosis.Recently,the application of ELRA for liver malignances has gained more attention.However,standardization of clinical practice norms and international consensus are still lacking.The prognostic impact in these oncologic patients also needs further evaluation.In this review,we summarized the principles and recent progresses on ELRA.

Ex vivo liver resection and auto-transplantation and special systemic therapy in perihilar cholangiocarcinoma treatment

This editorial contains comments on the article“Systematic sequential therapy for ex vivo liver resection and autotransplantation:A case report and review of li-terature”in the recent issue of World Journal of Gastrointestinal Surgery.It points out the actuality and importance of the article and focuses primarily on the role and place of ex vivo liver resection and autotransplantation(ELRAT)and systemic therapy,underlying molecular mechanisms for targeted therapy in perihilar cho-langiocarcinoma(pCCA)management.pCCA is a tough malignancy with a high proportion of advanced disease at the time of diagnosis.The only curative option is radical surgery.Surgical excision and reconstruction become extremely com-plicated and not always could be performed even in localized disease.On the other hand,ELRAT takes its place among surgical options for carefully selected pCCA patients.In advanced disease,systemic therapy becomes a viable option to prolong survival.This editorial describes current possibilities in chemotherapy and reveals underlying mechanisms and projections in targeted therapy with ki-nase inhibitors and immunotherapy in both palliative and adjuvant settings.Fi-broblast grow factor and fibroblast grow factor receptor,human epidermal grow-th factor receptor 2,isocitrate dehydrogenase,and protein kinase cAMP activated catalytic subunit alpha(PRKACA)and beta(PRKACB)pathways have been ac-tively investigated in CCA in last years.Several agents were introduced and approved by the Food and Drug Administration.They all demonstrated mean-ingful activity in CCA patients with no global change in outcomes.That is why every successfully treated patient counts,especially those with advanced disease.In conclusion,pCCA is still hard to treat due to late diagnosis and extremely complicated surgical options.ELRAT also brings some hope,but it could be performed in very carefully selected patients.Advanced disease requires systemic anticancer treatment,which is supposed to be individualized according to the genetic and molecular features of cancer cells.Targeted therapy in combination with chemo-immunotherapy could be effective in susceptible patients.

Dendritic cell-mimicking scaffolds for ex vivo T cell expansion

We propose an ex vivo T cell expansion system that mimics natural antigen-presenting cells(APCs)for adoptive cell therapy(ACT).Microfiber scaffolds coated with dendritic cell(DC)membrane replicate physicochemical properties of dendritic cells specific for T cell activation such as rapid recognition by T cells,long duration of T cell tethering,and DC-specific co-stimulatory cues.The DC membrane-coated scaffold is first surface-immobilized with T cell stimulatory ligands,anti-CD3(αCD3)and anti-CD28(αCD28)antibodies,followed by adsorption of releasable interleukin-2(IL-2).The scaffolds present both surface and soluble cues to T cells ex vivo in the same way that these cues are presented by natural APCs in vivo.We demonstrate that the DC-mimicking scaffold promotes greater polyclonal expansion of primary human T cells as compared toαCD3/αCD28-func-tionalized Dynabead.More importantly,major histocompatibility complex molecules derived from the DC membrane of the scaffold allow antigen-specific T cell expansion with target cell-specific killing ability.In addition,most of the expanded T cells(~97%)can be harvested from the scaffold by density gradient centri-fugation.Overall,the DC-mimicking scaffold offers a scalable,modular,and customizable platform for rapid expansion of highly functional T cells for ACT.

Systematic sequential therapy for ex vivo liver resection and autotransplantation: A case report and review of literature

BACKGROUND Perihilar cholangiocarcinoma(pCCA)is a highly malignant tumor arising from the biliary tree.Radical surgery is the only treatment offering a chance of long-term survival.However,limited by the tumor’s anatomic location and peri-vascular invasion,most patients lose the chance for curative treatment.Therefore,more methods to increase the resectability of tumors as well as to improve outcomes are needed.CASE SUMMARY A 68-year-old female patient had a hepatic hilar mass without obvious symptoms.Laboratory results showed hepatitis B positivity.Magnetic resonance imaging indicated that the mass(maximum diameter:41 mm)invaded the left and right branches of the main portal vein,as well as the middle,left and right hepatic veins;enlarged lymph nodes were also detected in the hilum.The patient was diagnosed with pCCA,and the clinical stage was determined to be T4N1M0(stage IIIC).Considering the tumor’s anatomic location and vascular invasion,systematic conversion therapy followed by ex vivo liver resection and autotrans-plantation(ELRA)was determined as personalized treatment for this patient.Our original systemic sequential therapeutic strategy(lenvatinib and tislelizumab in combination with gemcitabine and cisplatin)was successfully adopted as conversion therapy because she achieved partial response after three cycles of treatment,without severe toxicity.ELRA,anastomotic reconstruction of the middle hepatic vein,right hepatic vein,root of portal vein,inferior vena cava and right hepatic artery,and lymph node dissection were performed at one month after systemic therapy.Pathological and immunohistochemical examination confirmed the diagnosis of pCCA with lymph node metastasis.Although the middle hepatic vein was partially obstructed four months later,hepatic vein stent implantation successfully addressed this problem.The patient has survived for 22 mo after the diagnosis,with no evidence of recurrence or metastasis.CONCLUSION An effective therapeutic strategy for conversion therapy greatly increases the feasibility and efficiency of ELRA.

Complex inferior vena cava reconstruction during ex vivo liver resection and autotransplantation:A case report

BACKGROUND Ex vivo liver resection and autotransplantation(ELRA)is an essential approach for treating patients with end-stage hepatic alveolar echinococcosis(AE),and its surgical indications involve severe invasion of important hepatic vessels,which makes in vivo resection impossible.Revascularization is a major step in the process of ELRA,which is extremely challenging when the invaded vessels have huge defects.CASE SUMMARY Herein,we have reported the case of a 26-year-old patient with hepatic AE in an autologous liver graft who underwent complex inferior vena cava(IVC)reconstruction using disease-free IVC,autologous portal vein fragments,and umbilical vein within the ligamentum teres hepatis.The patient showed good surgical recovery without vascular-related complications during the long-term follow-up.CONCLUSION We reviewed three studies that have reported complex revascularization of the IVC.This case report and systematic review showed that the use of autologous perihepatic vessels prevents donor-area trauma,immune rejection,and other adverse reactions.When the blood vessel is severely invaded and a single vascular material cannot repair and reconstruct the defect,ELRA may provide a safe and feasible surgical approach,which has good prospects for clinical application.

THE EFFECT OF STEM CELL FACTOR, INTERLEUKIN-6 AND ERYTHROPOIETIN ON EXPANSION OF CD34^+ CELLS FROM HUMAN UMBILICAL CORD BLOOD

CD34+ cells from human umbilical cord blood were purified by Dynal beads M-450 CD34 immunoselection system and cultured in the presence of various cytokines alone or in combination, including stem cell factor (SCF), interleukin-6 (IL-6) and erythropoietin (EPO). The results revealed that: (D In methylcellulose culture, the plating efficiencies of purified cord blood CD34+ cells were much different when stimulated by various cytokines. IL-6 alone had the lowest colo-ny yield, while the combination of SCF, IL-6 and EPO had the highest yield. ② In the suspension culture, IL-6 alone or IL-6 + EPO had little expanding effect on cord blood CD34+ celis, the other cytokine combinations could expand cord blood CD34+ celis at different Ievels. Among them, the combination of SCF, IL-6 and EPO had the maximal expanding effect on cord blood CD34+ celis, the number of progenitor celis peaked at day 21, about 29-fold increase and nucleated celis increased approximately 3676-fold at day 28. The expanding effect of

The Cross-talk between ROS and p38MAPKα in the Ex Vivo Expanded Human Umbilical Cord Blood CD133^+ Cells

This study investigated the correlation between and compared the effects of reactive oxygen species（ROS） and p38 mitogen-activated protein kinase α（p38MAPKα） in the ex vivo expanded umbilical cord blood（hUCB） CD133＋ cells.hUCB CD133＋ cells were cultured in the hematopoietic stem cells（HSCs） culture medium with N-acetylcysteine（NAC,an anti-oxidant）,p38MAPKα-specific inhibitor（SB203580） or their combination.The levels of ROS and expression of phosphorylated p38MAPKα（p-p38） in CD133＋ cells were flow cytometrically detected.The efficacy of ex vivo expansion was evaluated by the density of CD133＋ cell sub-group colony-forming cells（CFC） and cobblestone area-forming cells（CAFC） assay.Our results showed decreased ROS levels in NAC,SB203580,and their combination treatment groups were almost 37%,48%,and 85%,respectively.Furthermore,SB203580 abrogated the activation of p38MAPKα more obviously than NAC.Moreover,the CD133＋ cells in SB203580 treatment group had a 21.93±1.36-fold increase,and 14.50±1.19-fold increase in NAC treatment group,but only 10.13±0.57-fold increase in control group.In addition,SB203580 treatment led a higher level increase in the number of CFU and CAFC than NAC did.These findings suggested that,in expanded CD133＋ cells,ROS activates p38MAPKα,which,in turn,induces ROS production,and p38MAPKα might be the most suitable regulator in ROS-p38MAPKα pathway for the promotion of HSCs ex vivo expansion.

Effect of Angiotensin Ⅱ on Cord Blood CD^(34+) Cells Expansion in vitro

In order to investigate the influence of angiotensin Ⅱ on hematopoietic system, CD34 + cells in cord blood were purified, and the effects of angiotensin Ⅱ in combination with various cytokines on their growth and differentiation were studied by cell culture in vitro. It was found that angiotensin Ⅱ in suspending medium could stimulate both BFU-E and CFU-GM expansion. The number of BFU-E and CFU-GM was increased with the increases of angiotensin Ⅱ concentrations during a certain range. In addition, the expansion fold of CFU-GM was increased from 2.3±0.8 times to 7.8±2.3 times when angiotensin Ⅱ was added in the presence of SCF+G-CSF+GM-CSF+IL-3 cytokines mixture. Similarly, the expansion fold of BFU-E was increased from 3.1±1.8 times to 9 2±2.3 times with angiotensin Ⅱ in the presence of SCF+EPO+TPO+IL-3. In the semi-solid medium, angiotensin Ⅱ could stimulate CFU-GM expansion but had no effect on the growth of BFU-E. In conclusion, angiotensin Ⅱ had some stimulating effects on cord blood hematopoietic progenitors expansion in vitro in the presence of other cytokines.

Ex vivo expansion of regulatory T cells for clinical applications against graft-versus-host disease in allogeneic hematopoietic stem cell transplantation

Objective To review the characteristics of regulatory T cells （Tregs） and ex vivo expansion of Tregs for treatment of graftversus-host disease （GVHD）.Data sources The data used in this review were retrieved from PubMed （1970-2013）.The terms ＂ex vivo expansion＂,＂regulatory T cell＂,and ＂graft-versus-host disease＂ were used for literature search.Study selection The publications about the characteristics of Tregs,ex vivo expansion of Tregs and clinical applications of Tregs against GVHD were identified,retrieved and reviewed.Results Tregs can be classified as natural Tregs （nTregs） and induced Tregs （iTregs）.Both subsets share most Treg features.Given their immunosuppressive property,Tregs have been tested for their capability of preventing GVHD.The bottleneck of Treg therapy is the limited numbers of naturally existing Tregs.To solve this problem,ex vivo expansion of nTregs or iTregs has been executed.The initial data indicate Treg therapy is effective in reducing GVHD without compromising graft-versus-leukemia （GVL）.Conclusion Ex vivo expansion of Tregs is a reliable way to prepare sufficient number of Tregs for management of GVHD.

Dynamic roles of angiopoietin-like proteins 1, 2, 3, 4, 6 and 7 in the survival and enhancement of ex vivo expansion of bone-marrow hematopoietic stem cells

Recent advances in hematopoietic stem cells(HSCs)expansion by growth factors including angiopoietin-like proteins(Angptls)have opened up the possibility to use HSCs in regenerative medicine.However,the unavailability of true in vitro HSCs expansion by these growth factors has limited the understanding of the cellular and molecular mechanism of HSCs expansion.Here,we report the functional role of mouse Angptls 1,2,3,4,6 and 7 and growth factors SCF,TPO,IGF-2 and FGF-1 on purified mouse bone-marrow(BM)Lineage-Sca-1+(Lin-Sca-1+)HSCs.The recombinant retroviral transduced-CHO-S cells that secrete Angptls in serum-free medium were used alone or in combination with growth factors(SCF,TPO,IGF-2 and FGF-1).None of the Angptls stimu-lated HSC proliferation,enhanced or inhibited HSCs colony formation,but they did support the survival of HSCs.By contrast,any of the six Angptls together with saturating levels of growth factors dramatically stimulated a 3-to 4.5-fold net expansion of HSCs compared to stimulation with a combination of those growth factors alone.These findings lead to an understanding of the basic function of Angptls on signaling pathways for the survival as well as expansion of HSCs in the bone marrow niche.

Ex vivo expansion of hematopoietic stem cells

Ex vivo expansion of hematopoietic stem cells(HSCs) would benefit clinical applications in several aspects, to improve patient survival, utilize cord blood stem cells for adult applications, and selectively propagate stem cell populations after genetic manipulation. In this review we summarize and discuss recent advances in the culture systems of mouse and human HSCs, which include stroma/HSC co-culture, continuous perfusion and fed-batch cultures, and those supplemented with extrinsic ligands, membrane transportable transcription factors, complement components, protein modification enzymes, metabolites, or small molecule chemicals. Some of the expansion systems have been tested in clinical trials. The optimal condition for ex vivo expansion of the primitive and functional human HSCs is still under development. An improved understanding of the mechanisms for HSC cell fate determination and the HSC culture characteristics will guide development of new strategies to overcome difficulties. In the future, development of a combination treatment regimen with agents that enhance self-renewal, block differentiation, and improve homing will be critical. Methods to enhance yields and lower cost during collection and processing should be employed. The employment of an efficient system for ex vivo expansion of HSCs will facilitate the further development of novel strategies for cell and gene therapies including genome editing.

Clinical Outcomes of Ex Vivo Liver Resection and Liver Autotransplantation for Hepatic Alveolar Echinococcosis

The effectiveness of liver autotransplantation for patients with partial hepatic alveolar echinococcosis was analyzed.We retrospectively studied 6 patients with hepatic alveolar echinococcosis who underwent liver autotransplantation in our hospital from 2008 to 2010.We also summarized the surgical indications of liver autotransplantation for hepatic alveolar echinococcosis and our experience in the management of postoperative complications of liver autotransplantation.Of 6 patients,5 achieved good curative results,and one died of multiple organ failure caused by portal vein thrombosis.Main complications included postoperative bleeding,bile leak and small-for-size liver graft syndrome.Liver autotransplantation offers a new approach to cure hepatic alveolar echinococcosis with non-resectable lesions.It could be the most effective method to cure intractable hepatic alveolar echinococcosis if correct handling in operation and proper prevention of complications are performed.But the long-term outcomes are still needed to be confirmed in longer follow-up.

Impelling force and current challenges by chemicals in somatic cell reprogramming and expansion beyond hepatocytes

In the field of regenerative medicine,generating numerous transplantable functional cells in the laboratory setting on a large scale is a major challenge.However,the in vitro maintenance and expansion of terminally differentiated cells are challenging because of the lack of specific environmental and intercellular signal stimulations,markedly hindering their therapeutic application.Remarkably,the generation of stem/progenitor cells or functional cells with effective proliferative potential is markedly in demand for disease modeling,cell-based transplantation,and drug discovery.Despite the potent genetic manipulation of transcription factors,integration-free chemically defined approaches for the conversion of somatic cell fate have garnered considerable attention in recent years.This review aims to summarize the progress thus far and discuss the advantages,limitations,and challenges of the impact of full chemicals on the stepwise reprogramming of pluripotency,direct lineage conversion,and direct lineage expansion on somatic cells.Owing to the current chemical-mediated induction,reprogrammed pluripotent stem cells with reproducibility difficulties,and direct lineage converted cells with marked functional deficiency,it is imperative to generate the desired cell types directly by chemically inducing their potent proliferation ability through a lineagecommitted progenitor state,while upholding the maturation and engraftment capacity posttransplantation in vivo.Together with the comprehensive understanding of the mechanism of chemical drives,as well as the elucidation of specificity and commonalities,the precise manipulation of the expansion for diverse functional cell types could broaden the available cell sources and enhance the cellular function for clinical application in future.

Ex vivo liver resection followed by autotransplantation in radical resection of gastric cancer liver metastases:A case report

BACKGROUND Radical resection of gastric cancer liver metastases(GCLM)can increase the 5-year survival rate of GCLM patients.However,patients may lose the theoretical feasibility of surgery due to the critical location of liver metastasis in some cases.CASE SUMMARY A 29-year-old woman had a chief complaint of chronic abdominal pain for 1 year.Abdominal computed tomography and magnetic resonance imaging examinations suggested a mass of unknown pathological nature located between the first and second hila and the margin of the lower segment of the right lobe of the liver.The anterior wall of the gastric antrum was unevenly thickened.The diagnosis of(gastric antrum)intramucosal well-differentiated adenocarcinoma was histopathologically confirmed by puncture biopsy with gastroscopy guidance.She underwent radical resection(excision of both gastric tumors and ex vivo liver resection followed by autotransplantation simultaneously)followed by XELOX adjuvant chemotherapy.Without serious postoperative complications,the patient was successfully discharged on the 20th day after the operation.Pathological examination of the excised specimen indicated that gastrectomy with D2 lymph node dissection for primary gastric tumors and R0 resection for liver metastases were achieved.The resected mass was confirmed to be poorly differentiated gastric carcinoma(hepatoid adenocarcinoma with neuroendocrine differentiation)with liver metastases in segments VIII.No recurrence or metastasis within the liver was found during a 7.5-year follow-up review that began 1 mo after surgery.CONCLUSION Application of ex vivo liver resection followed by autotransplantation in radical resection for GCLM can help selected patients with intrahepatic metastases located in complex sites obtain a favorable clinical outcome.

Approaches to reconstruction of inferior vena cava by ex vivo liver resection and autotransplantation in 114 patients with hepatic alveolar echinococcosis

BACKGROUND Hepatic alveolar echinococcosis(AE)is most commonly found in retrohepatic inferior vena cava(RHIVC).Ex vivo liver resection and autotransplantation(ELRA)can better realize the radical resection of end-stage hepatic AE with severely compromised hepatocaval confluences,and reconstruction of the affected vessels.Currently,there is a scarcity of information regarding RHIVC reconstruction in ELRA.AIM To propose reasonable RHICV reconstruction strategies for ex vivo liver resection and autotransplantation.METHODS We retrospectively summarized the clinical data of 114 patients diagnosed with hepatic AE who treated by ELRA in our department.A total of 114 patients were divided into three groups according to the different reconstruction methods of RHIVC:Group A with original RHIVC being repaired and reconstructed(n=64),group B with RHIVC being replaced(n=43),and group C with RHIVC being resected without reconstruction(n=7).The clinical data of patients,including the operation time,anhepatic phase,intraoperative blood loss,complications and postoperative hospital stay,were analyzed and the patients were routinely followed up.The normally distributed continuous variables were expressed as means±SD,whereas the abnormally distributed ones were expressed as median and analyzed by analysis of variance.Survival curve was plotted by the Kaplan-Meier method.RESULTS All patients were routinely followed up for a median duration of 52(range,12-125)mo.The 30 d mortality rate was 7.0%(8/114)and 7 patients died within 90 d.Among all subjects,the inferior vena cava(IVC)-related complication rates were 17.5%(11/63)in group A and 16.3%(7/43)in group B.IVC stenosis was found in 12 patients(10.5%),whereas thrombus was formed in 6 patients(5.3%).Twenty-two patients had grade III or higher complications,with the complication rates being 17.2%,16.3%,and 57.1%in the three groups.The average postoperative hospital stay in the three groups was 32.3±19.8,26.7±18.2,and 51.3±29.4 d(P=0.03),respectively.CONCLUSION ELRA can be considered a safe and feasible option for end-stage hepatic AE patients with RHIVC infiltration.The RHIVC reconstruction methods should be selected appropriately depending on the defect degree of AE lesions in IVC lumen.The RHIVC resection without any reconstruction method should be considered with caution.

Ex vivo limb perfusion for traumatic amputation in military medicine

Background:Limb loss has a drastic impact on a patient’s life.Severe trauma to the extremities is common in current military conflicts.Among other aspects,"life before limb"damage control surgery hinders immediate replantation within the short post-traumatic timeframe,which is limited in part by the ischemic time for successful replantation.Ex vivo limb perfusion is currently being researched in animal models and shows promising results for its application in human limb replantation and allotransplantation.Presentation of the hypothesis:The current lack of replantation possibilities in military operations with high rates of amputation can be addressed with the development of a portable ex vivo limb perfusion device,as there are several opportunities present with the introduction of this technique on the horizon.We hypothesize that ex vivo limb perfusion will enable overcoming the critical ischemic time,provide surgical opportunities such as preparation of the stump and limb,allow for spare-part surgery,enable rigorous antibiotic treatment of the limb,reduce ischemiareperfusion injuries,enable a tissue function assessment before replantation,and enable the development of large limb transplant programs.Testing the hypothesis:Data from in vivo studies in porcine models are limited by the relatively short perfusion time of 24 h.In the military setting,notably longer perfusion times need to be realized.Therefore,future animal studies must focus especially on long-term perfusion,since this represents the military setting,considering the time for stabilization of the patient until evacuation to a tertiary treatment center.Implications of the hypothesis:The development and clinical introduction of ex vivo limb perfusion in the military setting could lead to a drastic reduction in the number of limb amputations among service members.Ex vivo limb perfusion enables replantation surgery in Role 4 facilities and changes the clinical setting from a highly urgent,lifethreatening situation to a highly methodical,well-prepared starting point for optimal treatment of the wounded service member.With its introduction,the principle of"life before limb"will change to"life before limb before elective replantation/allotransplantation after ex vivo limb perfusion".

Ex vivo 3D osteocyte network construction with primary murine bone cells

Osteocytes reside as three-dimensionally（3D） networked cells in the lacunocanalicular structure of bones and regulate bone and mineral homeostasis. Despite of their important regulatory roles, in vitro studies of osteocytes have been challenging because：（1） current cell lines do not sufficiently represent the phenotypic features of mature osteocytes and（2） primary cells rapidly differentiate to osteoblasts upon isolation. In this study, we used a 3D perfusion culture approach to：（1） construct the 3D cellular network of primary murine osteocytes by biomimetic assembly with microbeads and（2） reproduce ex vivo the phenotype of primary murine osteocytes, for the first time to our best knowledge. In order to enable 3D construction with a sufficient number of viable cells, we used a proliferated osteoblastic population of healthy cells outgrown from digested bone chips. The diameter of microbeads was controlled to：（1） distribute and entrap cells within the interstitial spaces between the microbeads and（2） maintain average cell-to-cell distance to be about 19 mm. The entrapped cells formed a 3D cellular network by extending and connecting their processes through openings between the microbeads. Also, with increasing culture time, the entrapped cells exhibited the characteristic gene expressions（SOST and FGF23） and nonproliferative behavior of mature osteocytes. In contrast, 2D-cultured cells continued their osteoblastic differentiation and proliferation. This 3D biomimetic approach is expected to provide a new means of：（1） studying flow-induced shear stress on the mechanotransduction function of primary osteocytes,（2） studying physiological functions of 3D-networked osteocytes with in vitro convenience,and（3） developing clinically relevant human bone disease models.