组织工程化膀胱平滑肌结构的体外构建

目的 利用组织工程方法在体外初步构建膀胱平滑肌结构.方法 置备膀胱脱细胞基质移植物(BAMG)并以HE和Masson染色进行支架材料评价.酶消化法获得膀胱平滑肌原代细胞,分别以免疫荧光和RT-PCR进行细胞鉴定.经过体外培养和扩增后,将P3代膀胱平滑肌细胞接种在BAMG上.结果 支架上的膀胱平滑肌细胞经过体外培养4周后形成多层结构,同时免疫组化检测α-Smooth Muscle Actin表达阳性.结论 运用组织工程方法能够在体外进行膀胱平滑肌结构的初步构建,为进一步膀胱组织工程修复奠定技术基础.     

组织工程化膀胱壁复层结构的体外构建

目的 探讨膀胱缺损组织工程修复方法.方法 机械法去除膀胱黏膜层、肌层和浆膜层,仅保留膀胱黏膜下层.经SDS、Trixon-100和三蒸水处理去除细胞结构,制备获得膀胱脱细胞基质移植物(BAMG)作为支架材料.取成年猪膀胱壁,机械法分离膀胱肌层和黏膜层.以酶消化法分别获取膀胱尿路上皮细胞和平滑肌细胞.常规细胞传代和扩增后,将膀胱平滑肌细胞和尿路上皮细胞分别接种在BAMG浆膜面和黏膜面.细胞-材料复合物体外培养4周并行组织学鉴定.结果 HE和Masson染色鉴定发现,BAMG以胶原成分为主,内部未见明显细胞残留.免疫荧光方法鉴定结果显示,膀胱尿路上皮细胞uroplakin Ⅱ阳性,膀胱平滑肌细胞α-平滑肌肌动蛋白阳性.体外培养4周后,BAMG浆膜面和黏膜面分别形成连续的复层结构尿路上皮层和膀胱平滑肌层,免疫组化显示平滑肌层α-平滑肌肌动蛋白表达阳性,尿路上皮层广谱角蛋白表达阳性.结论 以膀胱脱细胞材料作为支架,尿路上皮细胞和膀胱平滑肌细胞作为种子细胞,能够在体外构建形成包含膀胱黏膜层及平滑肌层的复层膀胱壁结构.细胞-材料复合物的体外构建将为体内膀胱缺损的组织工程修复提供实验依据.     

人舌黏膜上皮细胞体外构建组织工程尿道的初步研究

目的 探索人舌黏膜上皮细胞和脱细胞支架体外复合构建组织工程尿道的可行性.方法 前尿道狭窄患者10例,手术切取0.5 cm×0.8 cm大小舌黏膜组织,分离获得舌黏膜上皮细胞,AE1/AE3抗体行细胞免疫荧光鉴定.收集第3代上皮细胞,按1×107/ml密度分别接种于脱水BAMG支架、液体保存BAMG支架以及4层脱细胞基质(SIS)支架表面,体外培养7d后行HE及扫描电镜检测.结果 10例患者术后1个月未出现舌部相关并发症.14 d后原代舌黏膜上皮细胞融合达90％～95％并呈典型的“鹅卵石”状生长.第3代时增殖速度达顶峰,平均7d达到90％～95％融合,第4代开始细胞逐渐老化.HE及扫描电镜检测液体保存BAMG支架表面仅复合极少量细胞,脱水BAMG以及SIS支架表面可见明显多层上皮细胞覆盖,其中4层SIS支架内部可见局部细胞浸润性生长迹象 结论 人舌黏膜上皮细胞可以作为组织工程尿道上皮种子细胞来源之一,与脱水处理后的SIS和BAMG有很好的组织相容性和黏附能力,二者的有效复合可以构建适合尿道修复重建需要的组织工程替代材料.     

脂肪干细胞复合膀胱脱细胞基质促进膀胱再生的实验研究

目的 探讨脂肪干细胞(adipose-derived stem cell,ADSC)在兔的组织工程膀胱重建中的应用.方法 采用自体脂肪干细胞和膀胱脱细胞基质(bladder acellular matrix graft,BAMG)构建兔组织工程膀胱.应用酶消化法分离培养兔自体的ADSC,并进行流式细胞鉴定.对新鲜的兔膀胱组织进行脱细胞处理制得BAMG.将ADSC扩增后接种于BAMG表面并进行体外培养,获得组织工程膀胱.对24只雄性新西兰兔行40％ ～ 50％膀胱部分切除,分为实验组和对照组,每组12只.实验组采用自体培养的ADSC构建的组织工程膀胱对缺损膀胱进行修复,对照组采用单纯的BAMG进行修复.术后24周进行膀胱造影并取材观察组织修复再生情况. 结果 镜下观察ADSC贴壁后绝大多数为纺锤形,细胞之间以细丝拉网.流式细胞仪检测结果CD90、CD4、CD105、CD166、CD34表达均为阳性,CD106、CD45表达为阴性.ADSC种植于BAMG表面生长良好.修补24周后,对照组和实验组膀胱容量分别为术前的(69.33 ±5.05)％和(94.68±3.31)％.组织染色分析对照组显示上皮层正常,黏膜下纤维组织增生而肌层较薄;实验组则显示了与自体膀胱相似的正常的3层组织结构. 结论 采用ADSC和BAMG构建组织工程膀胱是理想的膀胱替代修补材料.     

应用组织工程口腔黏膜重建尿道的初步实验研究

目的 探讨兔口腔黏膜细胞的体外培养方法,并以其为种子细胞与异体膀胱黏膜下脱细胞基质(bladder acellular matrix graft,BAMG)复合,构建组织工程尿道. 方法 18只10周龄雄性新西兰大耳白兔,体重0.3～0.5 kg.取其中12只兔口腔黏膜组织,分离获得口腔黏膜细胞.将口腔黏膜细胞分别接种于有3T3细胞及无3T3细胞的培养皿进行培养,接种后第2天于倒置相差显微镜下观察细胞形态变化,绘制细胞生长曲线,观察生长增殖情况,并行免疫荧光组织化学染色鉴定.取余6只兔膀胱,经脱细胞处理制备BAMG,随机取1 cm×1 cm组织行HE染色,观察脱细胞效果.将接种于有3T3细胞培养皿培养获得的第2代口腔黏膜细胞种植于BAMG上,培养1周后,行HE染色及扫描电镜观察口腔黏膜细胞与BAMG的复合状况. 结果 倒置相差显微镜下观察,接种于有3T3细胞培养皿的口腔黏膜细胞可传至7～8代,其细胞形态均一,功能良好;无3T3细胞培养皿的口腔黏膜细胞形态多样,传至第2代时,即开始出现老化.细胞生长曲线显示,两种方法培养的口腔黏膜细胞均于第8天开始呈对数增长,第14天达峰值.接种于有3T3细胞培养皿的口腔黏膜细胞,培养至融合时所获得细胞数量明显多于接种于无3T3细胞培养皿的u腔黏膜细胞.两种方法培养的口腔黏膜细胞行免疫荧光染色,均呈绿色荧光.HE染色观察,BAMG经脱细胞处理后未见细胞,脱细胞效果良好.复合培养1周后,HE染色及扫描电镜观察口腔黏膜细胞与BAMG复合良好. 结论 兔口腔黏膜细胞可在体外成功培养、扩增,与同种异体BAMG复合后生长良好,为构建组织工程尿道提供了一种新的选择.     

聚羟基乙酸作为支架材料体外构建复层膀胱壁结构的探讨

目的：探讨聚羟基乙酸（polyglycolic acid,PGA）作为支架材料并复合成年猪膀胱平滑肌细胞和尿路上皮细胞,体外构建复层膀胱壁结构的可行性。方法：采用PGA作为支架材料。并以聚乳酸（polylact acid,PLA）塑形。以酶消化法分别获得猪膀胱尿路上皮细胞和平滑肌细胞,并体外扩增。以MTT法分别检测P3代膀胱平滑肌细胞（SMC）和尿路上皮细胞（UC）的体外增殖能力。将P3代膀胱平滑肌细胞先接种在支架上,再接种尿路上皮细胞。将细胞一材料复合物体外培养1～2周并行组织学鉴定。结果：酶消化法获得的膀胱平滑肌细胞和尿路上皮细胞经过体外培养仍具有较强的体外增殖能力。细胞材料复合物体外培养1周后形成分层排列结构,包括黏膜上皮层和膀胱平滑肌层。免疫组织化学显示平滑肌层α-Smooth Muscle Actin表达阳性,黏膜上皮层广谱角蛋白表达阳性。而细胞材料复合物继续体外培养至2周,细胞从支架上大量脱落。结论：PGA适合作为复层膀胱壁结构体外构建的支架材料。采用PGA作为支架材料并复合膀胱平滑肌细胞和尿路上皮细胞,可以体外构建出类似复层膀胱壁结构。细胞材料复合物体外培养1周左右较适合进行体内回植修复。     

组织工程舌黏膜构建的研究

目的 探讨舌黏膜细胞的体外培养方法,及其作为种子细胞与同种异体膀胱脱细胞移植物(BAMG)复合构建组织工程化黏膜的方法.方法 分离并获取雄性新西兰大耳白兔舌黏膜细胞进行培养,定期观察细胞形态变化及生长增殖情况.对体外培养的舌黏膜细胞进行广谱角蛋白抗体(AE1/AE3)免疫荧光染色鉴定;流式细胞仪检测第2代培养细胞的AE1/AE3阳性细胞百分比;细胞计数及噻唑蓝(MTT)比色法测定以判断细胞增殖能力.取同种异体兔膀胱经脱细胞处理制成BAMG,随机取小块组织行HE和Masson染色观察脱细胞效果,然后将第2代体外培养的舌黏膜细胞种植于BAMG上,通过HE染色及扫描电镜观察口腔黏膜细胞与BAMG的复合情况.结果 舌黏膜细胞形态均一有较强的增殖能力可传代至4～5代,传至第4代时开始出现老化,细胞贴壁及增殖能力均明显减弱.免疫荧光染色显示近100%体外培养的舌黏膜细胞AE1/AE3免疫荧光染色阳性.流式细胞仪检测结果表明第2代舌黏膜细胞AE1/AE3阳性细胞百分比大于96%.舌黏膜细胞传至第3代时可获得细胞数量在2×107以上.HE和扫描电镜观察均显示舌黏膜细胞和BAMG复合良好.结论 兔舌黏膜细胞可在体外成功培养、扩增;兔舌黏膜细胞与同种异体BAMG复合后生长良好.     

尿道移行上皮细胞与生物可降解尿道支架体外复合培养的研究

目的:研究体外培养的兔尿道上皮细胞在生物可降解性网状尿道支架上的贴附和生长增殖情况,观察其对尿道上皮细胞形态和功能的影响,利用组织工程技术培养种植细胞的尿道内支架.方法:应用机械分离与酶消化法分离培养兔尿道移行上皮细胞,并在体外行原代培养与扩增后制成细胞悬液,接种在网状尿道支架上,形成尿道移行上皮细胞-支架复合物.应用免疫组织化学、荧光染色法鉴定尿道上皮细胞及其活性,并用倒置显微镜、扫描电镜观察尿道上皮细胞在支架表面吸附与生长状态.结果:网状尿道支架具有良好的生物相容性,能使尿道移行上皮细胞增殖,不影响其活性.尿道移行上皮细胞在尿道支架上贴附生长良好,1～2天后完全贴壁,3～7天细胞生长增殖活跃,支架网眼内充满上皮细胞;长期培养仍保持尿道移行上皮细胞特性,扫描电镜可见上皮细胞与网状支架紧密贴附,适度伸展并有基质分泌.结论:网状尿道支架适合尿道移行上皮细胞黏附生长,可作为尿道组织工程的细胞载体,利用组织工程方法可获得适于移植尿道细胞的组织工程化尿道.     

小肠粘膜下层复合成骨诱导的骨髓基质干细胞体内成骨的实验研究

目的探讨利用组织工程方法,以小肠粘膜下层为支架材料复合成骨诱导后的骨髓基质干细胞构建骨组织的可行性。方法将取自兔骨髓中的骨髓基质干细胞经成骨诱导液诱导后,与经处理的猪小肠粘膜下层在体外共培养。1周后,将共培养的猪小肠粘膜下层埋置于无胸腺裸鼠皮下。分别在不同时间进行扫描电镜、透射电镜、组织学和免疫组织化学观察。结果体外培养时,见细胞与材料粘附良好,且分泌大量的细胞外基质,细胞分化、增殖活跃。大体观察植入体内的细胞-材料复合物,见颜色变白,组织硬度增加,组织学和电镜观察见有大量骨组织形成。免疫组化示细胞为具有分泌特异性骨钙蛋白的成骨细胞。结论骨髓基质干细胞经成骨诱导为成骨细胞后与小肠粘膜下层共培养,植入裸鼠体内后可形成骨组织,小肠粘膜下层是一种良好的骨组织工程支架材料。     

聚磷酸钙纤维／左旋聚乳酸软骨支架复合自体骨髓间充质干细胞构建组织工程化人工关节软骨

目的：应用组织工程学技术,体外初步构建组织工程化人工关节软骨。方法：制备三维多孔软骨支架材料CPP／PLLA,体外诱导兔MSCs向软骨细胞表型分化,免疫组织化学染色检测软骨特异性Ⅱ型胶原表达,将诱导细胞与软骨支架材料CPP／PLLA复合,体外培养构建人工关节软骨,1周后终止培养,扫描电镜观察组织工程化人工软骨的微观结构;同时将构建人工软骨移植于兔大腿皮下,3周后处死动物,甲苯胺蓝染色观察。结果：扫描电镜观察可见该复合材料CPP／PLLA为高孔隙率的网状、连通、微孔结构,微孔分布均匀,孔径大小为300～400μm之间;兔MSCs经体外软骨表型定向诱导后,Ⅱ型胶原免疫组化染色阳性。诱导后的MSCs可在支架材料内良好贴附生长,细胞被分泌的胶原基质包裹;从体内获取的培养物组织切片观察可见大量的软骨细胞生成,甲苯胺蓝染色阳性。结论：经软骨起源诱导后的MSCs与CPP／PLLA复合培养可以构建自体软骨移植的替代物,为应用软骨组织工程方法修复关节软骨缺损和功能重建提供一种新材料,具有较大的潜在应用价值。     

Ureteral gene transfer to porcine induced strictures using endourologic delivery of an adenoviral vector

PURPOSE: Direct gene transfer to the ureter is an attractive approach to prevent restenosis after endourologic management of ureteral strictures. We therefore assessed the rationale for adenovirus-mediated gene transfer in the ureter in vitro and in vivo using a porcine model. MATERIALS AND METHODS: Primary cultures of porcine ureteral epithelial and stromal cells were infected with an adenoviral solution carrying a nucleus-targeted beta-Galactosidase (beta-Gal) reporter gene (6.5 10(8) pfu/ml.). In addition, in order to mimic the human clinical situation, we have devised a model of thermally-induced stricture in porcine ureter which produced tight fibrotic stenosis within 8 days. Using a purposely designed channelled balloon catheter prototype, these strictures were endoscopically dilated and then instilled with the same beta-Gal adenoviral construction. RESULTS: Application of recombinant adenovirus harboring a nucleus-targeted beta-Gal reporter gene to cultured porcine urothelial and stromal cells resulted in high transduction efficiency of up to 99% and 84% respectively. Seven days after infection, X-Gal staining of the strictured ureters demonstrated transfection up to 2 mm. depth within the fibrosis, confirmed by polymerase chain reaction (PCR) analysis. Adjacent and distal spread of the virus was excluded by histochemistry (X-Gal staining) and PCR. CONCLUSION: This data represents the first report of adenovirus-mediated gene transfer to the ureter. It remained site specific by endourologic retrograde clinically applicable techniques.     

Mechanisms of thrombotic microangiopathy following xenogeneic hematopoietic progenitor cell transplantation.

INTRODUCTION: Attempts to induce tolerance though mixed hematopoietic chimerism in the discordant pig-to-baboon xenotransplantation model are sometimes complicated by a potentially fatal thrombotic microangiopathy in the recipient baboons. This state develops immediately after the infusion of porcine mobilized peripheral blood leukocytes, containing progenitor cells (PBPC). In our study, we examined the interaction of infused porcine PBPC with recipient platelets in vivo in baboons and investigated the underlying mechanisms using an in vitro model. METHODS: Two na?ve baboons and six baboons preconditioned with irradiation and immunosuppression that received porcine PBPC were evaluated in vivo. The interaction of porcine and baboon PBPC with baboon platelets was investigated by an in vitro platelet aggregation assay. Fresh and cryopreserved PBPC were evaluated as well as PBPC obtained from growth-factor mobilized and unmobilized pigs. Furthermore, cellular subsets of PBPC were assessed for potential to induce platelet aggregation. Immunohistochemical staining was performed on platelet-leukocyte aggregates and potential inhibition of aggregation with anti-P-selectin and anti-CD154 mAbs, or eptifibatide (a GPIIb/IIIa receptor antagonist), was tested. RESULTS: All baboons that received porcine PBPC rapidly developed marked thrombocytopenia (<20,000/microl), elevated serum lactate dehydrogenase (>1,500U/liter), schistocytosis, and platelet aggregates on blood smear. Three baboons died (two untreated and one preconditioned), and substantive platelet aggregates containing porcine leukocytes were observed in the microvasculature of lungs and kidneys. In vitro, porcine, but not baboon, PBPC induced aggregation of baboon platelets in a dose-dependent manner. Immunohistological examination of these aggregates confirmed the incorporation of porcine leukocytes. Cryopreserved PBPC caused less aggregation than fresh PBPC, and growth-factor-mobilized PBPC induced less aggregation than unmobilized PBPC. Aggregation was fully abrogated by the addition of eptifibatide, and modulated by anti-P-selectin and anti-CD154 monoclonal antibodies that recognize adhesion receptors on activated platelets. Purified fractions (granulocytes, CD2+, and CD- cells) of porcine PBPC did not initiate aggregation, whereas addition of exogenous porcine PBPC membranes (erythrocytes, dead cells, and/or platelets) to the purified fractions exacerbated the aggregation response. CONCLUSIONS: These data indicate that porcine PBPC mediate aggregation of baboon platelets. This process likely contributes to the thrombotic microangiopathy observed after PBPC transplantation in the pig-to-baboon model. Eptifibatide can fully abrogate platelet aggregation induced by porcine PBPC in vitro. Purification of the progenitor cells from porcine PBPC and/or treatment of baboons with eptifibatide may be beneficial.     

Resistance of established porcine islet xenografts to humoral rejection by hyperimmune sera.

BACKGROUND: Although preformed natural antibodies cause hyperacute rejection of primarily vascularized xenografts, tissue grafts such as skin or islets are revascularized by in-growth of host capillaries and therefore might be resistant to circulating antibodies. We examined the effect of hyperimmune serum and primed T cells on the survival of long-term porcine islet xenografts in diabetic nude mice. METHODS: Porcine islets were transplanted beneath the kidney capsule of streptozotocin-induced diabetic BALB/c athymic mice. Hyperimmune serum and sensitized splenocytes were prepared by repeated immunization of BALB/c mice with porcine lymph node cells. Splenic T cells were enriched by nylon wool column separation. Tissues were examined by immunohistology using murine- and porcine-specific monoclonal antibodies. RESULTS: Porcine islets survived in nude mice for > 100 days with high levels of circulating porcine C-peptide and maintenance of normoglycemia. Injection of the hyperimmune sera (IgG) into normoglycemic nude mice bearing porcine islets for > 70 days failed to induce rejection despite the continued presence of circulating anti-porcine cytotoxic antibody. Injection of sensitized T cells caused acute rejection of long-term (>140 days) porcine islets, whereas injection of naive T cells had no effect. Histologically, porcine islets removed from mice treated with hyperimmune serum showed no staining for IgG. Long-surviving porcine islet grafts showed strong staining for interleukin (IL)-10 and a lesser amount of IL-4 but no staining for IL-2 or interferon-gamma. Although fresh porcine islets were positive for swine leukocyte antigen class 1 antigen and intercellular adhesion molecule (ICAM)-1 but negative for mouse platelet endothelial cell adhesion molecule and ICAM-2, long-surviving porcine islets showed positive endothelial staining for mouse platelet endothelial cell adhesion molecule and ICAM-2. CONCLUSIONS: Established islet xenografts are resistant to hyperimmune serum as a result of a lack of target endothelial antigens, whereas they remain susceptible to rejection caused by primed T cells. Local production of Th2 cytokines may explain the inability of long-surviving islet xenografts to activate injected naive T cells.     

Human Peripheral Blood Lymphocyte-Reconstituted, Severe Combined Immunodeficient Mice as a Model for Porcine Islet Xenograft Rejection in Humans

Abstract: Previously we established human peripheral blood lymphocyte-reconstituted severe combined immunodeficiency (SCID) (hu-PBL-SCID) mice as a model for human islet allograft rejection. The function of xenografted hu-PBL was confirmed to reject human allois-lets in hu-PBL-SCID mice. In this study, we modified this model as a porcine islet xenograft to study porcine islet rejection in humans. Chimeric mice were used as the recipients of porcine islets to reveal the mechanisms of xenograft rejection in humans. SCID mice were reconstituted with 30 times 10⁶ of hu-PBL initially, and 10 times 10⁶ of antihuman CD3-primed PBL was injected intraperitoneally 2 days later as a booster. An additional booster injection provided greater possibility (86.7%, n = 15) of chimera establishment as well as a higher human immunoglobulin concentration in SCID mice than the single injection group. In an in vitro assay, sera from hu-PBL-SCID mice were found to recognize porcine islets by FACS staining. In an in vivo study, immunofluorescent analysis of a frozen section showed that human immunoglobulins adhered to the xenografted porcine islet under the kidney capsule of hu-PBL-SCID mice. Although no mouse immunoglobulins were detected on sections, mouse complement (C3) was shown to adhere to the xenografted porcine islet. Thus, hu-PBL-SCID mice provide a useful model for investigating the real-life situation of porcine islet xenograft rejection in humans.     

Isolation and Culture of β-Like Cells From Porcine Wirsung Duct

We sought to develop a protocol to isolate and culture porcine Wirsung duct cells in order to determine their potency to differentiate into insulin-expressing β-like cells. The porcine Wirsung duct isolated by a surgical microdissection was digested with collagenase P and trypsin to dissociate ductal cells. These elements were cultured in serum-free supplemented media: for 2 weeks. Thereafter the cells were exposed to varying concentrations of glucose (0, 5.6, 17.8, and 25 mmol/L) to induce a β-like phenotype, as identified by immunohistochemical staining. Cell growth proceeded slowly for the first 2 weeks of culture. After glucose induction for 2 weeks, they formed pancreatic islet-like structures. These cells were stained for the pancreatic ductal cell marker cytokeratin-19 (CK-19) and the pancreatic endocrine markers insulin and glucagon. After the second week, 90% of cells were positive for CK-19. Up to 20.1% of the cells in pancreatic 3-dimensional structures induced by 17.8 mmol/L glucose were positive for insulin, and <3.2%, for glucagon. The positive ratio of immunoreactive staining was dependent on the glucose concentration; 17.8 mmol/L glucose effectively stimulated insulin- and glucagon-secreting cells. We concluded that porcine Wirsung duct cells were capable of proliferation with the potential to differentiate toward β cells upon glucose induction in vitro.     

Dissecting the instant blood-mediated inflammatory reaction in islet xenotransplantation

Abstract: Background: A massive destruction of transplanted tissue occurs immediately following transplantation of pancreatic islets from pig to non‐human primates. The detrimental instant blood‐mediated inflammatory reaction (IBMIR), triggered by the porcine islets, is a likely explanation for this tissue loss. This reaction may also be responsible for mediating an adaptive immune response in the recipient that requires a heavy immunosuppressive regimen. Materials and methods: Low molecular weight dextran sulfate (LMW‐DS) and the complement inhibitor Compstatin were used in a combination of in vitro and in vivo studies designed to dissect the xenogeneic IBMIR in a non‐human primate model of pancreatic islet transplantation. Adult porcine islets (10 000 IEQs/kg) were transplanted intraportally into three pairs of cynomolgus monkeys that had been treated with LMW‐DS or heparin (control), and the effects on the IBMIR were characterized. Porcine islets were also incubated in human blood plasma in vitro to assess complement inhibition by LMW‐DS and Compstatin. Results: Morphological scoring and immunohistochemical staining revealed that the severe islet destruction and macrophage, neutrophilic granulocyte, and T‐cell infiltration observed in the control (heparin‐treated) animals were abrogated in the LMW‐DS‐treated monkeys. Both coagulation and complement activation were significantly reduced in monkeys treated with LMW‐DS, but IgM and complement fragments were still found on the islet surface. This residual complement activation could be inhibited by Compstatin in vitro. Conclusions: The xenogeneic IBMIR in this non‐human primate model is characterized by an immediate binding of antibodies that triggers deleterious complement activation and a subsequent clotting reaction that leads to further complement activation. The effectiveness of LMW‐DS (in vivo and in vitro) and Compstatin (in vitro) in inhibiting this IBMIR provides the basis for a protocol that can be used to abrogate the IBMIR in pig‐human clinical islet transplantation.     

Live encapsulated porcine islets from a type 1 diabetic patient 9.5 yr after xenotransplantation

BACKGROUND: The long-term viability and function of transplanted encapsulated neonatal porcine islets was examined in a diabetic patient. METHODS AND RESULTS: A 41-yr-old Caucasian male with type 1 diabetes for 18 yr was given an intraperitoneal transplant of alginate-encapsulated porcine islets at the dose of 15,000 islet equivalents (IEQs)/kg bodyweight (total dose 1,305,000 IEQs) via laparoscopy. By 12 weeks following the transplant, his insulin dose was significantly reduced by 30% (P = 0.0001 by multiple regression tests) from 53 units daily prior to transplant. The insulin dose returned to the pre-transplant level at week 49. Improvement in glycaemic control continued as reflected by total glycated haemoglobin of 7.8% at 14 months from a pre-transplant level of 9.3%. Urinary porcine C-peptide peaked at 4 months (9.5 ng/ml) and remained detectable for 11 months (0.6 ng/ml). The patient was followed as part of a long-term microbiologic monitoring programme which subsequently showed no evidence of porcine viral or retroviral infection. At laparoscopy 9.5 yr after transplantation, abundant nodules were seen throughout the peritoneum. Biopsies of the nodules showed opacified capsules containing cell clusters that stained as live cells under fluorescence microscopy. Immunohistology noted sparse insulin and moderate glucagon staining cells. The retrieved capsules produced a small amount of insulin when placed in high glucose concentrations in vitro. An oral glucose tolerance test induced a small rise in serum of immuno-reactive insulin, identified as porcine by reversed phase high pressure liquid chromatography. CONCLUSION: This form of xenotransplantation treatment has the potential for sustained benefit in human type 1 diabetics.