梯度血清递减体外培养人脐带间充质干细胞

背景：人脐带间充质干细胞的扩增与培养条件密切相关,而含体积分数10%~20%胎牛血清的培养基可促进细胞生长。 目的：建立梯度血清递减扩增培养脐带间充质干细胞的培养技术。 方法：采用胶原酶Ⅱ消化分离获得脐带间充质干细胞悬液,并通过贴壁培养进行纯化,细胞贴壁后采用梯度血清递减方法,即第1代80%含血清培养基,20%无血清培养基;第2代50%含血清培养基,50%无血清培养基;第3代20%含血清培养基,80%无血清培养基;第4代100%无血清培养基。另一种传代中始终采用含体积分数10%胎牛血清的α-MEM完全培养液。用流式细胞仪检测细胞的表面标记,进行成骨诱导试验,同时与经典的含10%胎牛血清的α-MEM培养体系进行比较。 结果与结论：梯度血清递减培养法与经典α-MEM培养体系所获得的细胞在扩增能力、细胞形态、免疫表型等方面相似。细胞在两种培养体系中均能保持良好的分化潜能。但梯度血清浓度法培养间充质干细胞可使用更少量胎牛血清,提高临床应用安全性。     

人脐血中分离培养扩增内皮集落形成细胞及其生物相容性

背景：内皮集落形成细胞是内皮祖细胞的一种亚型,体外培养扩增及其在组织工程中的应用尚不明确。 目的：探索从人脐血中分离培养扩增内皮集落形成细胞并观察其生物相容性。 方法：采用密度梯度离心法从人脐血中分离单个核细胞,接种于Ⅰ型鼠尾胶原包被的培养板上,采用内皮祖细胞专用EGM-2培养基诱导培养,早期传代后扩增,免疫荧光鉴定细胞,并检测其体外成血管和增殖能力,与纳米羟基磷灰石/磷酸三钙材料复合培养后的生物相容性。 结果与结论：脐血来源内皮集落形成细胞呈铺路石样集落生长,吞噬Dil-Ac-LDL并结合FITC-UEA-1,表达CD31,vWF,KDR和Tie-2,早期传代后拥有较强增殖潜能,可在体外大量扩增,在体外基质胶上可形成闭合管腔样结构,复合纳米羟基磷灰石/磷酸三钙材料后生物学特性不受影响。提示可从脐血中分离并体外大量扩增内皮集落形成细胞。     

Endothelial colony forming cells generated from cryopreserved peripheral blood mononuclear cells

Derivation of endothelial colony forming cells (ECFCs) from peripheral blood mononuclear cells (PBMCs) is a technique that could provide access to donor endothelial cells to study donor endothelium/recipient immune cells interactions. The success rate of ECFC colony formation from cryopreserved PBMCs has not been reported. We used biobanked PBMCs and studied the yield of ECFC generation. Endothelial phenotype was confirmed with CD31, CD146, CD309, CD34, CD14 and CD11c staining by flow cytometry and VE-cadherin, von Willebrand factor and Dil-Ac-LDL by fluorescent microscopy. Functionality was tested by endothelial cell tube-based formation assay. The success rate of ECFC generation was 28%. Freezing time was not a predictor of ECFC generation while a shorter time on dialysis and living transplant were significant determinants. These data suggest that it is possible to generate ECFCs from cryopreserved PBMCs, which is a potentially useful option for the longitudinal assessment of alloimmune response in transplantation.     

Umbilical cord tissue-derived mesenchymal stem cells grow best under GMP-compliant culture conditions and maintain their phenotypic and functional properties

Mesenchymal stem cells (MSCs) are fibroblast-like multipotent stem cells that can differentiate into cell types of mesenchymal origin. Because of their immune properties and differentiation, potential MSCs are discussed for the use in tissue regeneration and tolerance induction in transplant medicine. This cell type can easily be obtained from the umbilical cord tissue (UCMSC) without medical intervention. Standard culture conditions include fetal bovine serum (FBS) which may not be approved for clinical settings. Here, we analyzed the phenotypic and functional properties of UCMSC under xeno-free (XF, containing GMP-certified human serum) and serum-free (SF) culture conditions in comparison with standard UCMSC cultures. Phenotypically, UCMSC showed no differences in the expression of mesenchymal markers or differentiation capacity. Functionally, XF and SF-cultured UCMSC have comparable adipogenic, osteogenic, and endothelial differentiation potential. Interestingly, the UCMSC-mediated suppression of T cell proliferation in an allogeneic mixed lymphocyte reaction (MLR) is more effective in XF and SF media than in standard FBS-containing cultures. Regarding the mechanism of action of MLR suppression, transwell experiments revealed that in neither UCMSC culture a direct cell-cell contact is necessary for inhibiting T cell proliferation, and that the major effector molecule is prostaglandin E₂ (PGE₂). Taken together, GMP-compliant growth media qualify for long-term cultures of UCMSC which is important for a future clinical study design in regenerative and transplant medicine.     

血清替代培养法使血清外泌体干扰最小化

外泌体是细胞之间及细胞和细胞外基质之间信号交流的途径之一。近年来,对外泌体的研究呈现显著增长的态势。在体外细胞培养系统中,为保证细胞正常的生理状态,血清是培养液中必须添加的成分。随之而来的问题是,由于血清中含有大量外泌体,其数量数倍于研究中需要进行针对性研究的特定外泌体,从而对特定外泌体的定量和分析造成了干扰。为了去除培养液血清中的外泌体,很多研究者采用差速离心法、密度梯度离心法等进行处理。该研究通过流式细胞分析对外泌体定量后发现,加入2%血清的培养液在进行差速离心处理后,外泌体数量减少了30.6%;而加入5%的血清培养液,外泌体数量下降了32.4%。由此可见,差速离心处理对不同浓度血清的培养液结果类似,只能去除约30%的血清外泌体,留下约70%的血清外泌体,仍然会对实验数据造成干扰。因此,在人脐静脉内皮细胞体外培养系统中,该研究探索了一种新型血清替代培养法,即以无生物成分的血清替代物XerumFree替代培养液中的大部分血清,经过对一系列配比进行探索发现,当XerumFree与胎牛血清FBS体积比为15:1时,细胞既可以正常生长,又可以分泌外泌体,而背景中外泌体干扰程度降低到2%,从而较好地解决了外源性外泌体形成的背景干扰问题,为体外研究外泌体提供了明确的参考方案。     

Peptide-grafted poly(ethylene glycol) hydrogels support dynamic adhesion of endothelial progenitor cells

This study investigated the dynamic adhesion of endothelial progenitor cells (EPCs) to peptide-grafted poly(ethylene glycol) diacrylate (PEGDA) hydrogels and determined the relative ability of RGDS, REDV and YIGSRG peptides to reduce the velocity of EPC rolling. Circulating EPCs are key mediators of endothelium repair and have been shown to accelerate re-endothelialization, which is important in reducing the incidence of restenosis following stent placement and occlusion of small diameter vascular grafts. However, to exploit these capabilities for tissue engineering applications, more knowledge is needed about EPC binding to the vascular wall under shear and, in particular, whether the incorporation of peptide ligands into biomaterials can support the process of EPC rolling or maintain EPC adhesion. This study specifically examined one type of EPCs endothelial colony forming cells (ECFCs), based on their ability to be expanded in culture and differentiate into mature endothelial cells. The amount of grafted PEG–peptide was shown to be dependent on the concentration of PEG–peptide grafting solution photopolymerized onto the hydrogel surface. The ECFC strength of adhesion on PEG–RDGS grafted hydrogels exceeded 350 dyn cm^?2 for 85% of adherent cells. PEG–RGDS grafted hydrogels supported ECFC rolling, whereas ECFC velocity on the negative control PEG–RGES grafted hydrogels and on the “blank slate” PEGDA hydrogels was substantially higher than the cutoff velocity for cell rolling. The ECFC rolling velocity on PEG–RDGS grafted hydrogels depended on the shear rate; as shear rate was increased from 20 s^?1 to 120 s^?1, ECFC rolling velocity increased from 103 ± 3 μm s^?1 to 741 ± 28 μm s^?1. REDV and YIGSRG, which are known to preferentially support endothelial cell adhesion, also supported ECFC rolling. Interestingly, the rolling velocity of ECFCs on PEG–REDV grafted hydrogels was significantly lower than on PEG–YIGSRG or on PEG–RGDS grafted hydrogels. Understanding the dynamic adhesion of ECFCs to peptide-grafted hydrogels is the first step towards understanding the similarities and differences of EPCs from mature endothelial cells and improving the ability to sequester EPCs to biomaterial surfaces in order to promote intravascular re-endothelialization.     

适合人肌母细胞的无血清培养基

背景：肌母细胞体外培养大多使用添加胎牛血清培养基,存在很多不足,开发无血清培养基,更加稳定、安全、经济,有广泛的应用前景。 目的：初步探索适合人肌母细胞培养的无血清培养基。 方法：配制含胎牛血清培养基 (DMEM/F12 1∶1添加胰岛素样生长因子1、碱性成纤维细胞生长因子和体积分数20%胎牛血清),间充质干细胞无血清培养基组(无血清培养基、2%血清替代物、2 mmol/L L-谷氨酰胺,人脐带间充质干细胞,细胞纯度大于99%,UltraCULTURETM添加血清替代物,谷氨酰胺)。自制无血清培养基组(GibcoTM添加胰岛素样生长因子1,碱性成纤维细胞生长因子,谷氨酰胺)。3种培养基分别对人肌母细胞分别进行培养,观察肌母细胞的形态、免疫组织化学的鉴定,计算克隆的形成率,绘制细胞生长的曲线,MTT检测肌母细胞的活性,比较3种培养基培养肌母细胞增殖、分化的差异。 结果与结论：各组细胞形态上无明显差异;各组免疫组织化学鉴定肌母细胞纯度均达99%;含胎牛血清培养基组和自制无血清培养基组克隆形成率显著高于间充质干细胞无血清培养基组(P < 0.01),含胎牛血清培养基组克隆形成率显著高于自制无血清培养基组(P < 0.01);含胎牛血清培养基组与自制无血清培养基组细胞数远高于间充质干细胞无血清培养基组;自制无血清培养基组细胞活性显著高于含胎牛血清培养基组和间充质干细胞无血清培养基组(P < 0.01)。自制无血清培养基组可有效用于肌母细胞的培养,在促进肌母细胞早期贴壁、增殖上还需进一步优化。  中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程      

Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction

Adult human bone marrow-derived stem cells, having the ability to differentiate into cells of multiple lineages, have been isolated and propagated by varied protocols, including positive (CD105(+))/negative (CD45(-)GlyA(-)) selection with immunomagnetic beads, or direct plating into selective culture media. Each substratum-adherent cell population was subjected to a systematic analysis of their cell surface markers and differentiation potential. In the initial stages of culture, each cell population proliferated slowly, reaching confluence in 10-14 days. Adherent cells proliferated at similar rates whether cultured in serum-free medium supplemented with basic fibroblast growth factor, medium containing 2% fetal bovine serum (FBS) supplemented with epidermal growth factor and platelet-derived growth factor, or medium containing 10% FBS alone. Cell surface marker analysis revealed that more than 95% of the cells were positive for CD105/endoglin, a putative mesenchymal stem cell marker, and negative for CD34, CD31, and CD133, markers of hematopoietic, endothelial, and neural stem cells, respectively, regardless of cell isolation and propagation method. CD44 expression was variable, apparently dependent on serum concentration. Functional similarity of the stem cell populations was also observed, with each different cell population expressing the cell type-specific markers beta-tubulin, type II collagen, and desmin, and demonstrating endothelial tube formation when cultured under conditions favoring neural, cartilage, muscle, and endothelial cell differentiation, respectively. On the basis of these data, adult human bone marrow-derived stem cells cultured in adherent monolayer are virtually indistinguishable, both physically and functionally, regardless of the method of isolation or proliferative expansion.     

IL‐7 and immobilized Kit‐ligand stimulate serum‐ and stromal cell‐free cultures of precursor B‐cell lines and clones

Long‐term proliferating, D_HJ_H‐rearranged mouse precursor B‐cell lines have previously been established in serum‐ and IL‐7‐containing media from fetal liver, but not from bone marrow. Serum and stromal cells expose these pre‐B cells to undefined factors, hampering accurate analyses of ligand‐dependent signaling, which controls pre‐B cell proliferation, survival, residence and migration. Here, we describe a novel serum‐free, stromal cell‐free culture system, which allows us to establish and maintain pre‐B cells not only from fetal liver, but also from bone marrow with practically identical efficiencies in proliferation, cloning and differentiation. Surprisingly, recombinant kit‐ligand, also called stem cell factor, produced as a kit‐ligand‐Fc fusion protein, suffices to replace stromal cells and serum, provided that it is presented to cultured pre‐B cells in an optimal density in plate‐bound, insolubilized, potentially crosslinking form. Additional recombinant CXCL12 and fibronectin have a minor influence on the establishment and maintenance of pre‐B cell lines and clones from fetal liver, but are necessary to establish such cell lines from bone marrow.     

Fetal bovine serum contains an inhibitor of interleukin-1

The keratinocyte cell line COLO-16 constitutively produces factors with interleukin-1 (IL-1) activity including IL-1 alpha and IL-1 beta. IL-1 activity assayed by thymocyte proliferation from cell supernatants was 20-50% less if cells were maintained in media containing 10% fetal bovine serum (FBS) compared to media without serum 24 h prior to harvest. The increased IL-1 activity in supernatants from cells in serum free media was not due to increased cellular levels of IL-1 alpha or IL-1 beta mRNA. Similarly, IL-1 activity recovered from conditioned supernatants of COS cells transfected with expression vectors containing IL-1 beta cDNA was approximately 22-45% less in cells grown in 20% FBS medium compared to similar cultures grown for 3 days post transfection in 1% FBS. When serial dilutions of recombinant IL-1 were made in buffer containing 10% FBS and assayed by a thymocyte proliferation method, a 30-50% decrease in activity was observed. IL-1 activity was also measured by its ability to induce prostaglandin E2 synthesis by fibroblasts. When COS conditioned supernatants were applied to fibroblast cultures there was 30% less prostaglandin E2 activity from fibroblasts treated with COS supernatants containing 20% FBS, compared to supernatants containing 1% FBS. The inhibitor molecule was partially purified by gel filtration and found to have a molecular weight of approximately 85,000. The presence of FBS in cell-conditioned media significantly reduces the sensitivity of IL-1 detection by bioassay techniques.     

Hyaluronic Acid hydrogels support cord-like structures from endothelial colony-forming cells

The generation of functional vascular networks has the potential to improve treatment for vascular diseases and to facilitate successful organ transplantation. Endothelial colony-forming cells (ECFCs) have robust proliferative potential and can form vascular networks in vivo. ECFCs are recruited from a bone marrow niche to the site of vascularization, where cues from the extracellular matrix instigate vascular morphogenesis. Although this process has been elucidated using natural matrix, little is known about vascular morphogenesis by ECFCs in synthetic matrix, a xeno-free scaffold that can provide a more controllable and clinically relevant alternative for regenerative medicine. We sought to study hyaluronic acid (HA) hydrogels as three-dimensional scaffolds for capillary-like structure formation from ECFCs, and to determine the crucial parameters needed to design such synthetic scaffolds. We found that ECFCs express HA-specific receptors and that vascular endothelial growth factor stimulates hyaluronidase expression in ECFCs. Using a well-defined and controllable three-dimensional HA culture system, we were able to decouple the effect of matrix viscoelasticity from changes in adhesion peptide density. We determined that decreasing matrix viscoelasticity, which corresponds to a loose ultrastructure, significantly increases ECFC vascular tube length and area, and that the effect of local delivery of vascular endothelial growth factor within the hydrogel depends on the makeup of the synthetic environment. Collectively, these results set forth initial design criteria that need to be considered in developing vascularized tissue constructs.     

AKT蛋白激酶活化对弥漫性大B细胞淋巴瘤细胞生物学行为影响的体外研究

目的观察AKT及磷酸化AKT（pAKT,即AKT呈活化状态））蛋白激酶在三个弥漫性大B细胞淋巴瘤（DLBCL）细胞株中的表达状态,并探讨其与DLBCL细胞生物学行为的关系以及P13K抑制剂LY294002对该瘤细胞生长的影响。方法采用Western blot检测DLBCL细胞株ly1、ly8、ly10在有血清和无血清培养条件下AKT和pAKT的表达状态;用PBK抑制剂LY294002分别作用于三株细胞,观察pAKT水平的改变;用流式细胞术结合碘化丙啶（PI）单染分析细胞周期、Annexin V-异硫氢酸荧光素（FITC）染色检测细胞凋亡、BrdU法观察细胞增殖状态的改变。结果三株DLBCL细胞株在不同的培养条件下均显示有pAKT,在10％FBS培养组ly8和ly10之pAKT水平高于无血清培养组;而ly1相反,在无血清培养组pAKT略高于有血清培养组。在有血清培养组,LY294002可明显降低三细胞株细胞的pAKT水平,并显示S期细胞明显减少（P〈0．05）,G0-G1期细胞增加,增殖细胞比例显著降低（P〈0．05）,以ly8、ly10细胞尤为明显;但凋亡细胞无明显增加（P〉0．05）。结论AKT活化与DLBCL细胞周期和细胞增殖密切相关;与细胞凋亡无显著相关;LY294002可通过降低DLBCL细胞的pAKT水平继而抑制瘤细胞生长。     

Enhancement of lymphocyte proliferation assays by use of serum-free medium.

Fetal bovine serum (FBS)-supplemented cell culture medium has been the standard medium used in assays of murine lymphocyte proliferation. While FBS allows cell growth and viability, it requires screening and contains uncharacterized elements which may yield inconsistent results from batch to batch. It also may include growth factors and immunologically reactive proteins that obscure assays for specific responses because of high background proliferation. Recently, chemically defined serum-free media have become available for human lymphocyte culture. We compared several of these media against FBS-supplemented medium and found that one of them, AIM V (Gibco), produced a marked increase in the ratio of positive to background counts in [3H]thymidine incorporation assays of antigen-specific proliferation. Similar results were obtained when responses to mitogenic lectins were examined. AIM V supported proliferation of lymphocytes in a variety of haplotypes, it supported MHC-specific proliferation in response to soluble antigens in a manner consistent with previous reports, and it supported proliferation in response to alloantigen as displayed in the mixed lymphocyte culture. A high ratio of positive to background counts was consistently observed. The results indicate that this serum-free medium enhances the analytic power of proliferation assays.     

Differences in the effect on neural stem cells of fetal bovine serum in substrate-coated and soluble form

The influence of fetal bovine serum (FBS) adsorbed to poly(ethylene-co-vinyl alcohol) (EVAL) and polyvinyl alcohol (PVA) substrates (coated FBS) and FBS present in the culture medium (soluble FBS) on the behavior of embryonic rat cerebral cortical neural stem cells was studied at neurosphere level. When both coated FBS and soluble FBS were not present in the culture system, the fate and behavior of neurospheres were mediated mainly by the substrates used. When neurospheres were cultured either on FBS-coated EVAL or FBS-coated PVA substrates in the serum-free medium, the most striking morphological characteristic of neurospheres was that these neurosphere-forming cells attached and were induced to differentiate into process-bearing cell phenotypes predominantly; however, the differentiated cell phenotypes were dissimilar on these two substrates. On the contrary, when neurospheres were cultured in the medium containing 10% FBS, the neurosphere-forming cells were induced into protoplasmic cells typically but no difference in differentiated cell phenotypes on EVAL and PVA substrates was observed. Interestingly, instead of promoting process outgrowth under serum-free medium condition, coated FBS enhanced migration of differentiated protoplasmic cells when soluble FBS were present. These results inform that the substrates, coated serum, and soluble serum within the culture environment together can significantly alter cell behavior and morphological differentiation and will therefore be an important clue for the development of biomaterials to regulate the potential of the CNS neural stem cells.     

Enhancement of hybridoma production by medium supplemented with murine ascitic fluid

We have greatly improved the yield after cell fusion of antigen-specific monoclonal antibody-secreting murine hybridomas by substitution of Sp2/0 ascites for fetal bovine serum (FBS) in the culture medium. As a medium supplement for established cultures, Sp2/0 ascites at 2.5% in Dulbecco's modified Eagle's medium (DMEM) provided growth characteristics similar to media containing 10% or 20% FBS. All culture parameters associated with hybridoma fusion experiments, however, were advantageously affected in ascites-supplemented cultures. Clonal growth of hybridomas from the single cell stage was enhanced at least two-fold over 20% FBS-supplemented medium. Following fusion, both the number of colonies and hybridoma growth rates were substantially increased for ascites-containing cultures. Most importantly, the number of antigen-specific antibody-secreting hybridomas was increased in Sp2/0 ascites supplemented cultures, five-fold in the eight fusion experiments presented here. This improved performance compared to FBS-supplemented medium is reproducible from lot to lot of ascites.     

The influence of serum-free culture conditions on skeletal muscle differentiation in a tissue-engineered model

The influence of differentiation medium (DM) components on C2C12 murine myoblast differentiation has only been studied in monolayer cultures. Serum-free formulations have been applied that omit the use of sera with unknown composition. The goal of the present study was to compare the influence of serum-free media on C2C12 differentiation in 3-dimensional tissue-engineered muscle constructs. Myoblast proliferation and differentiation in media containing Ultroser G (DMU), insulin-like growth factor (IGF)-I (DMI), or both (DMUI) were compared with those induced by more-traditional media containing horse serum (HS) or horse serum and IGF-I (HSI). Effects of the applied media were assessed from gross construct morphology, total protein content, creatine kinase activity, and tissue viability. Addition of IGF-I (HSI) to the standard DM (HS) improved myoblast differentiation in muscle constructs. Even better results were obtained using DMU and DMUI culture conditions. DMI could not induce differentiation or maintain cell viability. Serum-free culture medium supplemented with DMU or DMUI accelerates and improves myoblast differentiation in engineered muscle tissue better than the gold standard HS.     

无血清培养体系原代培养脐带间充质干细胞

背景：以含血清培养基培养的脐带间充质干细胞,存在着进一步应用的障碍。 目的：建立无血清培养体系原代培养脐带间充质干细胞。 方法：取脐带华通氏胶(Wharton’s Jelly),采用机械法将组织胶切碎,1-3 mm3大小,分别培养到含胎牛血清完全培养液中以及无血清培养液中。培养第11,14,17天收获细胞并进行相关检测。在符合2006年制定的干细胞最低评估标准的基础上,以集落形成单元指标评估何种培养体系能获得较多高质量的原代细胞。 结果与结论：无血清培养体系相对于经典的含血清的培养体系而言,细胞生长速度更快。另外,培养第11天,集落形成实验表明无血清培养体系下能获得更多的集落。因此,无血清培养体系可以保持间充质干细胞的特性,为替代含血清培养体系进行脐带间充质干细胞原代培养提供了可能。     

脐带间充质干细胞在不同培养体系条件下的生物学特性

背景：体外培养的脐带间充质干细胞在不同培养体系下生长状态差异显著,因此选取一种更适合的培养基相当必要。 目的：对比观察人脐带间充质干细胞在3种培养基中的生长增殖情况,并检测细胞免疫表型以及间充质干细胞的诱导分化能力。 方法：在无菌条件下用贴块法收获人脐带间充质干细胞,用T75培养瓶培养传代后,取第3代脐带间充质干细胞分别种入到含体积分数为5%胎牛血清的DMEM/F12培养基、含体积分数为10%胎牛血清的DMEM/F12培养基和Mesen PRO RSTM培养基中,培养第1,3,5,7天进行细胞计数,绘制细胞生长曲线。采用流式细胞仪分析第3代脐带间充质干细胞免疫表型,并检测其成骨及成脂肪诱导分化能力。 结果与结论：培养出的第3代人脐带间充质干细胞高表达CD44、CD73、CD90、CD105,不表达CD29、CD31、CD34、HLA-DR。经成脂诱导后,油红O染色可见胞浆中有大量红色小脂滴;成骨诱导后,茜素红染色后镜下可见成骨样细胞团,说明脐带间充质干细胞在体外具有多向分化的潜能。在倒置显微镜下观察可见含体积分数为10%胎牛血清的DMEM/F12培养基中的细胞集落密集,形态均匀,而其他2种培养基中的细胞集落密集程度和形态都不如前者好。在培养传代细胞时,可优先选择含体积分数为10%胎牛血清的DMEM/F12培养基。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：