| Backgrounds:Islet transplantation can not only improve glycemic control in type1diabetes, butalso avoid hypoglycemia events following intensive insulin therapy，and it is also capableof achieving normoglycemia along with the prevention or even reversal of certainsecondary diabetic complications. About90percent of clinical islet transplantations havebeen performed by infusion into the liver through the portal vein. Although the liver sitehas been extremely well characterized, it remains suboptimal, as many of the islets are lostduring or shortly after transplantation, and the liver cannot sustain the long-time survivalof the islets. The interaction with blood to minimize activation of complement andcoagulation cascades known as the instant blood-mediated inflammatory reaction was alsoan unfavorable factor. Thus many natural implantation sites have been proposed for islettransplantation, including the vascular of the lung, liver, and spleen, within tissues ororgans, such as pancreas, subcutaneous, muscle, bone, kidney capsule, omental pouch, andthe immune privileged sites, like brain, thymus or testis. But all of these naturaltransplantation sites have problems when they were explored to clinical application.However, a new era of chamber devices provide opportunities for clinical therapeutic advance. Our laboratory has established an in vivo tissue engineering chamber model inprevious study. The chamber was made by silicone, and it was hollow and perforated.Following implanted subcutaneously, tissue fluid accumulated in the chamber spacethrough the hole that drilled on the surface. A variety of growth factors were detected inthe tissue fluid. And the tissue fluid can sustain cell survival and proliferation of severalkinds of cells in vitro. In addition, tissue engineered cartilage can be constructed throughchondrocytes/scaffold complexes in the silicone chamber. These results demonstrated thatthe in vivo chamber model not only constructed a certain space, but also provided tissuefluid which sustained cell survival and growth. Therefore we thought that the in vivochamber device could be used as alternative implantation site for endocrine celltransplantation, especially for the islet transplantation given that this kind of cellfunctioned through response to local environment glucose.Objective:⑴To establish the standard protocol for isolation and purification of mouse islet cellsin our laboratory.⑵To explore whether the tissue fluid aspirated from the in vivo chambercould sustained viability and function of islet cells in vitro.⑶To explore the feasibility ofthe in vivo silicone chamber space as an alternative islet transplantation site.⑷Toinvestigate the glycemic control following islet transplantation to the silicone chamber indiabetic mice.Methods:⑴Islet isolation: C57BL/6J mice islets were isolated through injection ofcollagenase P (1.3U/ml) to the common bile duct. Pancreatic islets were purified throughdensity gradient purification and hand picking.①DTZ staining was used to identify theislet. Islet count from a single pancreas was performed.②glucose-stimulated insulinsecretion (GSIS) was performed to evaluate the endocrine function of isolated islets. Theglucose stimulation index (SI) is the ratio of stimulated-to-basal insulin secretion.⑵Invitro experiments:①The silicone chamber was implanted subcutaneously of the mouseback. Tissue fluid was collected through a regular aspiration.②Islet cells were cultured intissue liquid and RPMI1640media containing different glucose concentration for2weeks. AO-PI staining was used to assess the viability of the cells. Glucose stimulated insulinrelease was performed to evaluate the function of cultured islet cells.⑶Feasibility study:①Relationship between blood glucose and tissue fluid glucose. After intraperitonealglucose injection, glucose levels were analyzed at0,15,30,60,90,120min of the bloodand tissue fluid respectively.②Glycemic control of insulin from the chamber space. Thechange of blood glucose was analyzed of different insulin administration routes (inerchamber space, subcutaneously, intraperitoneal).⑷In vivo study:①Type1diabetes micewere induced through a single intraperitoneal injection of streptozotocin at200g/kg bodyweight.②Silicone chambers were implanted subcutaneously2weeks prior to islettransplantion in mice.③Mouse islets were transplanted into the chamber space or underthe renal capsular. Non-fasting blood glucose was measured after transplantation.Intraperitoneal glucose tolerance test was performed at5th weeks. Tissues were harvestedfor HE staining and immunohistochemistry staining.⑸Statistical analysis: Statisticanalysis was performed by Prism5.0soft ware. Data are reported as mean±standarddeviation (SD) for all observations. To determine the significances of differences betweenmeans, for more than two groups one-way ANOVA and Tukey’s Multiple Comparison Testwere applied. ANOVA for randomized block design and the area under the curve wascomputed using the trapezoid rule by Prism5.0soft ware. A P value≤0.05was consideredsignificant.Results:⑴Islets were successfully isolated and purified.①purer islets were obtainedthrough density gradient purification and hand picking.②The total yield of a singlepancreas was145±27.5.③The glucose stimulation index was4.04±0.9of fresh isolatedislets.⑵Tissue fluid is a suitable media to maintain islet mass, morphology, andfunction during in vitro culture.①Tissue fluid accumulated in mouse in vivo siliconchamber containing5.5mM glucose.②After in vitro cultured for two weeks, lessprotruding from the relatively smooth rounded surface was observed of islets in tissuefluid compared with cultured in RPMI1640media.③The viability of islets after2-weekculture in low-glucose tissue fluid, high-glucose tissue fluid, high-glucose and low-glucose RPMI1640media was91%,84%,76%,63%respectively.④The stimulationindex in each group was3.81±0.72,2.72±0.63,2.63±0.81,1.33±0.79respectively. Bettermorphology and function maintenance of isolated mouse islets were obtained culturing intissue fluid than in RPMI1640media. Low-glucose tissue fluid was better thanhigh-glucose tissue fluid, while high-glucose RPMI1640media was better thanLow-glucose RPMI1640media.⑶Glucose level in the tissue fluid tracks the bloodglucose concentration closely, but negligible, time lag, especially when the bloodglucose is sharply increased in a very short time, while there was a route for systemicinsulin delivery from the chamber space to regulate glucose metabolism.①Thesuccess rate of the diabetic mice was81%induced by STZ.②Before glucose tolerance,glucose concentration in blood and tissue fluid was3.9±1.3mmol/L,3±1.1mmol/Lrespectively, there was no statistical difference (P>0.05). At15min after glucose tolerance,blood glucose significantly increased to17.2±2.3mmol/L, while the glucoseconcentration in tissue fluid was7.8±1.9mmol/L(P<0.05). Since then, glucose level inthe tissue fluid tracks the blood glucose concentration closely.③Insulin injected into thechamber space was as effective as insulin injected subcutaneously and intraperitoneally.⑷Islets transplanted in the chamber space could reversal diabetic mice.①Positiveinsulin and glucogon immunochistoemistry staining demonstrated the survival andfunction of islet cells of both chamber and renal sub-capsular graft groups.②Non-fastingblood glucose analysis revealed that islets transplanted in the chamber space couldreversal diabetic mice.③During IPGTT, the glucose change profile of islet chamber graftwas similar to that of renal sub-capsular and normal control groups.④During0-15minafter glucose tolerance, the total plasma insulin was lower in the islet chamber graft groupthan that of renal sub-capsular and normal control groups. Compared to sub-capsule group,islet chamber group displayed a38%and15%decrease in plasma insulin AUC0-15andAUC0-30, respectively, but comparable insulin AUC30-120and insulin AUC0-120.⑤At15minafter glucose tolerance, There was significant difference in glucose levels between bloodand tissue fluid. There was no significant difference in glucose levels between blood andtissue fluid at all the other time point since then. Blood glucose peaked at30min after glucose tolerance, while glucose in tissue fluid also peaked at this time point. There wasno significant difference in tissue fluid glucose levels between islet chamber graft groupand diabetic control group, while the insulin levels in tissue fluid was significantlyincreased between the two group（P<0.05）.Conclusion:⑴Mouse islets isolation and and purification is a complex procedures. Every detailshould be considered.⑵Islet cells were cultured by tissue fluid for the first time. Theresults demonstrated that better morphology and function of islets within tissues fluid than10%FBS RPMI1640media (11mM glucose). Tissue fluid was more close to the physicalenvironment of islets. Culturing islets in tissue fluid is a good model to study thephysiological and pathological changes of pancreatic islet cells in vivo.⑶The studydemonstrated the relationship between the blood glucose and tissue fluid glucose in adiffusion chamber for the first time. Glucose level in the tissue fluid tracks the bloodglucose concentration closely, but negligible, time lag, especially when the blood glucoseis sharply increased in a very short time, while there was a route for systemic insulindelivery from the chamber space.⑷The study demonstrated the difference between thevascularized islets and non-vascularized islets in the regulation of glucose metabolism.Response to the blood glucose of vascularized islets is more sensitive than the non-vascularized islets. The reason was that tissue fluid reflects blood glucose levels after abrief, but negligible, time lag. But there was no delay in insulin function of differentdrainage.⑸the in vivo silicone chamber can be used as an alternative site for islettransplantation, and it has the applicability in the clinical setting. |
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