大鼠骨髓间充质干细胞的分离与培养——贴壁分离和消化控制相结合可否为简便高效的技术方法

目的：对骨髓间充质干细胞的培养一般采用流式细胞仪分离法及密度梯度离心法和免疫磁珠分离法，本实验观察了采用贴壁分离和消化控制相结合方法分离纯化的可行性。方法：实验于2004—09／12在锦州医学院附属第一医院外科实验室完成。选取6—8周龄的SD大鼠，雌雄不限，从其股骨、胫骨中取材，在无菌条件下操作，用剪刀分别剪去股骨胫骨的两端，暴露骨髓腔，用含有体积分数为0．15的胎牛血清Dulbecco改良培养基从骨髓腔中冲出骨髓于平皿中，用含有体积分数为0．1的胎牛血清的Dulbecco改良培养基培养，用消化控制及贴壁分离法分离纯化骨髓间充质干细胞，并观察其生长状态，测定骨髓间充质干细胞的生长曲线，分裂指数，贴壁率，用免疫细胞化学方法对骨髓间充质干细胞进行表型分析。结果：原代培养的骨髓间充质干细胞呈椭圆型、短梭型、长梭型、多角型等。纯化、扩增后骨髓间充质干细胞呈均匀一致的长梭型，第6代前生长性状稳定，增殖能力强。传代周期为7d，每次传代时细胞活力测定均&;gt;97％，传代后24h内贴壁率99％。免疫细胞化学检测第3代细胞均匀表达CD54(ICAM-1)、纤维连接蛋白。结论：实验建立了一种可行的大鼠骨髓间充质干细胞取材、纯化、扩增方法。培养的骨髓间充质干细胞纯度高，细胞活力强，生物学性状稳定。本实验通过贴壁分离和消化控制相结合的培养方法具有操作简便，条件要求低等优点，是目前一种比较理想的分离纯化方法。     

兔骨髓间充质干细胞的分离、培养与鉴定

背景：目前对骨髓间充质干细胞常用的分离方法有密度梯度离心法、贴壁筛选分离法。目的：联合应用密度梯度离心法和贴壁筛选分离法体外分离培养、扩增兔骨髓间充质干细胞，并对其进行鉴定。设计、时间及地点：对比观察的细胞学实验，于2007-10／2008-03在上海市第六人民医院中心实验室完成。材料：2月龄新西兰纯种大耳白兔6只用于骨髓间充质干细胞取材与原代培养，1．073kg／L的Percoll分离液。方法：实验采用Percol分离液利用密度梯度离心法及结合贴壁分离筛选法来分离、纯化骨髓间充质干细胞，在采用密度梯度离心法得到骨髓间充质干细胞后，经贴壁培养及反复换液纯化骨髓间充质干细胞。分别取第3，5，7，9代骨髓间充质干细胞，行细胞计数，绘制细胞生长曲线。主要观察指标：倒置显微镜下观察原代及传代细胞的形态、生长情况。采用CD44及CD34抗体进行间接免疫荧光标记鉴定培养的干细胞。CD44染色呈阳性，CD34染色呈阴性，说明所提取、纯化的细胞是骨髓间充质干细胞。结果：增殖传代的骨髓间充质干细胞呈长梭形均匀分布生长，形态比原代培养的细胞更均匀，细胞生长旺盛、增殖迅速，胞核明显，核仁清晰，核浆比例大，细胞形态均匀，平行排列呈螺旋状或漩涡状，传代至第5代时无明显变化。随传代次数的增加，细胞增殖能力逐渐下降，第3～5代细胞增殖能力强。所分离培养的细胞均表达CD44，不表达CD34。结论：在体外采用密度梯度离心及贴壁培养法可获得高纯度的兔骨髓间充质干细胞。     

大鼠骨髓间充质干细胞体外分离培养与CM-DiI荧光标记

背景：目前,对骨髓间充质干细胞的分离、纯化和扩增还没有统一的、标准化的方法。CM-DiI作为荧光标记物稳定、可靠、标记率高、标记简便。 目的：建立SD大鼠骨髓间充质干细胞体外分离培养及标记的方法。 方法：取2只体质量50-100 g雄性SD大鼠,无菌条件下采集双侧股骨、胫骨骨髓,用全骨髓贴壁分离法和密度梯度离心法培养出原代骨髓间充质干细胞,通过及时、反复传代对细胞进行扩增纯化,在体外用荧光活性染料CM-DiI标记第3代骨髓间充质干细胞后作为供体细胞来源。 结果与结论：用全骨髓贴壁分离法和密度梯度离心法两种方法均能成功体外分离培养骨髓间充质干细胞,经流式细胞仪分析,培养出的细胞CD34阳性率为17.5%,CD44阳性率为97.9%、CD90阳性率为91%,与骨髓间充质干细胞表面抗原一致。但培养出的细胞数量全骨髓贴壁分离法明显多于密度梯度离心法,两种方法培养骨髓间充质干细胞的细胞活力和增殖能力无明显差异。CM-DiI能够成功荧光标记骨髓间充质干细胞, CM-DiI作为荧光标记物稳定、可靠、标记率高、标记简便。     

全骨髓贴壁并差速传代分离纯化大鼠骨髓间充质干细胞:与密度梯度离心法的比较

背景:目前国内外分离纯化骨髓间充质干细胞有两种主要方法;密度梯度离心法及全骨髓贴壁法,前者步骤较复杂,后者简单易操作,但纯化效果不理想.目的:在全骨髓贴壁分离骨髓间充质干细胞基础上,并用差速传代消化法,建立大鼠骨髓间充质干细胞体外分离培养纯化方法.方法:全骨髓贴壁培养法分离并差速消化传代大鼠骨髓间充质干细胞,利用间充质干细胞在消化传代过程中较其他骨髓细胞消化悬浮速度快,以及贴壁快的特点,代替密度梯度离心操作来分离纯化间充质干细胞,对其形态学特征进行观察,并与密度梯度离心法比较两种分离培养法的细胞生长增殖情况;观察碱性磷酸酶及油红染色情况,验证骨髓间充质干细胞的分化能力;检测细胞表面标记物,验证免疫特性及检测其纯度.结果与结论:全骨髓贴壁培养法分离并差速传代大鼠骨髓间充质干细胞,流式细胞鉴定、成骨成脂肪培养结果显示其细胞免疫特性、纯度、分化能力与密度梯度离心法无显著差异,但细胞活力,增殖能力有明显提高.     

豚鼠骨髓间充质干细胞的分离培养及成骨诱导

背景:利用骨髓间充质干细胞的取材灵活性及快捷性,对已掌握的培养技术及成骨诱导进一步探索性研究。目的:通过建立豚鼠骨髓间充质干细胞的体外分离培养法,探讨豚鼠骨髓间充质干细胞表型特征以及多项分化潜能。方法:利用贴壁培养法分离纯化豚鼠骨髓间充质干细胞,传代扩增,流式细胞分析检测细胞表面分子CD29、CD44、CD45的表达。分别采用成骨诱导培养液和成脂诱导培养液定向诱导骨髓间充质干细胞向脂肪细胞、成骨细胞分化。结果与结论:原代分离的骨髓间充质干细胞在接种后96h贴壁,细胞形态为椭圆形,多角形及短梭形,8d时细胞呈长梭形并达到90%单层融合。经传代扩增,细胞进一步纯化,细胞形态为均一的长梭形并呈漩涡状排列,而且生长速率加快。流式细胞检测CD29、CD44阳性率分别为95.7%和65.7%。不同诱导剂定向诱导后,经油红O、茜素红S、碱性磷酸酶染色、免疫组织化学Ι型胶原酶鉴定,P3代骨髓间充质干细胞分别向脂肪细胞及成骨细胞分化。结果表明,通过贴壁筛选方法,体外分离培养的豚鼠骨髓间充质干细胞具有很强的增殖能力,并保持稳定的表型特征及多向分化潜能。     

全骨髓法体外培养分离大鼠骨髓间充质干细胞及PKH26标记

背景：要获得动物实验需要的标记大鼠骨髓间充质干细胞,体外培养、扩增和示踪已成为实验的关键环节。目的：采用全骨髓培养分离大鼠骨髓间充质干细胞,以及PKH26对其体外标记,建立一种方便、实用的分离培养并示踪骨髓间充质干细胞的方法。方法：通过全骨髓培养分离法纯化大鼠骨髓间充质干细胞。经传代扩增,细胞进一步纯化。取第3代大鼠骨髓间充质干细胞按PKH26标记程序进行标记后培养,荧光显微镜下观察标记后细胞生长状态、萤光强度变化和传代培养效果。利用四唑盐比色法测定标记后骨髓间充质干细胞的生长曲线。结果与结论：全骨髓培养分离法能成功获得纯度高的骨髓间充质干细胞,用PKH26标记后的骨髓间充质干细胞呈红色荧光,体外连续传代培养3代后,细胞荧光强度逐渐减弱。PKH26标记骨髓间充质干细胞的生长形态、生长活力不发生改变。结果证实全骨髓培养分离法简便易行,能获取较高纯度的生长增殖状态良好的骨髓间充质干细胞,PKH26荧光标记大鼠骨髓间充质干细胞是一种有效、实用的方法。     

人骨髓间充质干细胞的贴壁分离与体外培养

目的:验证贴壁方式分离人骨髓间充质干细胞,并进行体外扩增培养的可行性。方法:实验于2006-02/12在解放军济南军区总医院脊髓修复科完成。①实验材料:骨髓来源于解放军济南军区总医院脊髓修复科收治的脊髓完全性损伤患者,对本实验知情同意。基础培养液由含体积分数为0.15胎牛血清和低糖α-MEM配置。②实验方法:无菌条件下髂后上棘穿刺抽取骨髓组织6mL,进行细胞培养,观察细胞生长情况,待细胞融合成片、长满培养瓶底部后,用质量浓度为2.5g/L的胰蛋白酶流过所有细胞表面。倒置显微镜下观察细胞变圆、部分脱壁后,立即加入有血清培养液终止消化。③实验评估:取第3代生长状态良好的细胞,胰蛋白酶消化制成细胞悬液,接种,以细胞数为纵坐标,时间为横坐标,绘制细胞生长曲线。同时每隔2h进行细胞贴壁率检测。结果:①骨髓间充质干细胞的形态学观察:倒置显微镜下,骨髓间充质干细胞接种1d即贴壁,去除悬浮细胞后继续培养3d贴壁细胞开始增殖,伸展为椭圆型、短梭型、多角型及不规则型等。至14d细胞密集在集落中心,基本铺满瓶底,第3～5代细胞呈均匀一致的长梭型,排列成旋涡状或放射状。②骨髓间充质干细胞的生长曲线:细胞传代后3d内处于潜伏期,3d后进入生长期,7d后进入平台期。③骨髓间充质干细胞的贴壁率:随着培养时间的延长,骨髓间充质干细胞贴壁率逐渐升高。传代后2,4,6,8,10,12,14,16,18,20h细胞贴壁率分别为(20.20±0.25)%,(33.00±0.29)%,(46.50±0.32)%,(69.20±0.30)%,(76.60±0.34)%,(86.50±0.27)%,(90.30±0.20)%,(96.10±0.28)%,(98.50±0.12)%,(99.00±0.07)%。结论:贴壁法分离骨髓间充质干细胞操作简便,经体外扩增培养后细胞增殖活性强,传代周期为7d,是比较理想的骨髓间充质干细胞培养方法。     

大鼠骨髓间充质干细胞体外分离及对原发性骨质疏松性骨折的实验研究

目的用全骨髓贴壁分离法分离培养骨髓间充质干细胞,通过建立原发性骨质疏松性骨折模型,探讨骨髓间充质干细胞对骨质疏松性骨折愈合情况。方法无菌条件下采集双侧股骨、胫骨骨髓,用全骨髓贴壁分离法培养出原代骨髓间充质干细胞,选取11周雌性SD大鼠60只,卵巢切除术(OVX)建立骨质疏松模型后,均做双侧骨折模型,随机分成对照组(OVX骨折不处理组)、生理盐水组(OVX骨折+生理盐水组)和细胞治疗组(OVX骨折+骨髓间充质干细胞治疗组),术后于第3天、第4、8周分别进行X-光扫描。结果全骨髓贴壁法成功分离出骨髓间充质干细胞,经流式细胞仪分析,培养出来的细胞与骨髓间充质干细胞表面抗原一致。X-光结果表明,细胞治疗组相比于其它组能加快骨质疏松性骨折愈合。结论体外分离大鼠骨髓间充质干细胞对原发性骨质疏松性骨折愈合有促进作用。     

贴壁法体外分离培养大鼠骨髓间充质干细胞的效果验证

背景:骨髓中的间充质干细胞含量不高,且随着年龄增加或体质衰弱,骨髓间充质干细胞的数量会逐渐减少.目的:验证贴壁法分离培养大鼠骨髓间充质干细胞的效果.方法:大鼠麻醉后取双侧股骨和胫骨,剪去骨骺端,暴露骨髓腔,用含小牛血清的DMEM培养基冲洗骨髓腔,收集骨髓细胞,反复吹打制成单细胞悬液,接种后置于37 ℃、体积分数为5%的CO_2培养箱内孵育,24 h后全量换液,以后每周全量换液1次,筛选易贴壁但贴壁不牢的细胞进行传代培养.观察细胞形态,绘制细胞生长曲线,流式细胞仪及免疫细胞化学染色鉴定骨髓间充质干细胞表面标志的表达.结果与结论:培养24 h后细胞能够贴壁生长,呈梭形或三角形;第二三天贴壁细胞迅速增殖;培养15 d左右出现致密的贴壁细胞层,呈漩涡状生长或成簇生长.细胞在接种后2 d进入对数生长期,12 d左右进入平台期,约15 d细胞可铺满瓶底.分离培养的大鼠骨髓间充质干细胞CD90和CD54均呈阳性表达.结果验证了采用贴壁法可在体外成功分离培养大鼠骨髓间充质干细胞,操作简单,造成污染的环节和机会较少,不需离心,可以更好的保持细胞活性.     

人骨髓间充质干细胞和汗腺细胞的体外分离与培养

背景：体外研究人骨髓间充质干细胞和汗腺细胞的分离培养与鉴定,可为探讨骨髓间充质干细胞再生汗腺的可行性打下基础。目的：探寻在体外分离培养骨髓间充质干细胞和汗腺细胞的有效方法。方法：采用直接贴壁法从成人骨髓中体外分离培养骨髓间充质干细胞,并进行扩增和鉴定。采用胶原酶消化法从人全层无烧伤皮肤中分离汗腺细胞,并进行扩增和鉴定。结果与结论：倒置显微镜下见分离培养的骨髓间充质干细胞呈梭形,折光性强,免疫细胞化学染色显示细胞表达CD29、CD105,高表达CD44,不表达造血干细胞表面标志CD34和CD45。汗腺细胞呈扁平多角形,表达汗腺细胞表面标志细胞角蛋白7,8,18,19和癌胚抗原。说明直接贴壁法分离培养骨髓间充质干细胞和胶原酶消化法分离培养汗腺细胞是可行的。     

Effects of separation and separation with supplemental stroking in BALb/c infant mice

The purpose of this pilot study was to investigate selected stress, immune, and growth consequences of maternal separation and separation with supplemental stroking in neonatal BALB/c infant mice and their dams. Three groups of 5 litters each (7 pups per litter) were studied. Control litters were undisturbed. Separated litters experienced 3 h of daily maternal deprivation on postnatal days 6 to 10. Separated/stroked litters were separated also, but for 2 h, which was then followed by 1 h of stroking with a wet paintbrush to simulate maternal tactile stimulation. After the experimental period, all animals were returned to the nest and left undisturbed for 5 additional days. One pup from each litter was sacrificed on postnatal days 6, 8, 10, and 15. Spleens and thymuses were removed, weighed, and homogenized for cell sorting, cytokine analysis, and proliferation studies. Blood was drawn for corticosterone levels and hematocrit. Hematocrits and thymus weights were lower in separated mice, suggesting decreased growth and protein synthesis. Separated/stroked pups had increased splenic proliferation responses to conconavalin A and phytohemagglutinin at day 15. Separated dams' proliferative response to ConA was lower than control dams at day 15. Day 15 decreases in thymic CD8 cells occurred in pups, with an increased thymic H:S ratio in separated pups. CD90 cells were higher at day 15 in separated/stroked pups as were CD25s at day 10 in spleen and thymus. However, gene expression of cytokines was not measurable in spleen and thymic cells, with the exception of gamma-IFN in separated/stroked animals. Pooled organ homogenates were used in this preliminary work, and further studies are needed to more precisely analyze the stress, immune, and growth effects of these interventions.     

应用干细胞分离仪分离脐血与传统手工分离法的效果比较

目的:脐血处理的关键问题是提高干细胞的回收率及实现处理过程的标准化和可重复化,实验对此进行探讨,比较干细胞分离仪与传统羟乙基淀粉手工法分离脐血的效果。方法:实验于2006-12/2007-05在广州医学院附属市一人民医院完成。①脐血来源:39份脐血采自广州医学院附属市一人命医院妇产科健康顺产新生儿脐带,产妇均知情同意。随机数字表法分为仪器分离组17份、手工分离组22份。②实验方法:仪器分离组收集脐血称质量,计算体积,在开始处理前20min缓慢加入相当于20%脐血体积的60g/L羟乙基淀粉。仪器分离组按仪器要求自动分离,分离终体积20mL。手工分离组50g离心5min,压浆板压出全部血浆以及18mL红细胞移至无菌空袋,500g离心13min,自动压浆板压出血浆,保留20mL终体积样本。③实验评估:采用全自动计数仪进行检测有核细胞(白细胞)、红细胞数量。流式细胞仪分析CD34 含量。结果:采用干细胞分离仪处理浓缩脐血,有核细胞回收率为(89.7±3.4)%,CD34 细胞回收率为(98.8±5.1)%,红细胞去除率为(55.2±16.7)%,均比手工分离组分离效果好,差异有显著性意义(P<0.05或0.01)。同时,仪器分离组有核细胞回收率、CD34 细胞回收率的标准差均明显低于手工分离组(3.4vs.15.3;5.1vs.10.3)。结论:相比传统的羟乙基淀粉手工分离法,干细胞分离仪脐血分离浓缩效果理想,且结果标准误小,数据稳定。     

Current status of granulocyte separation and transfusion

Granulocyte substitution therapy is of definitive clinical value when correctly indicated and consequently performed. Comparing the different techniques for leucocyte separation, the combination of continuous flow centrifugation (CFC) and filtration (FL) permits the highest yield of granulocytes. As yet, all available methods imply a rather high degree of inconvenience and a considerable rate of side effects for the (normal) donor which cannot easily be justified from a medical point of view. On the other hand, CFC and especially FL have a great potential for further improvement. Hence, higher yields and less donor affection are expectable. Granulocyte procurement is a great challenge to existing transfusion centres requiring their future engagement and expansion in this new and important field.     

Superhydrophilic fluorinated polyarylate membranes via in situ photocopolymerization and microphase separation for efficient separation of oil-in-water emulsion

A superhydrophilic modified fluorinated polyarylate membrane with high tensile strength was prepared by a combination of in situ photocopolymerization and microphase separation. The as-prepared membrane was successfully utilized for oil-in-water emulsion separation with high separation efficiency and high flux. Furthermore, the membrane displayed excellent antifouling performance and recyclability for long-term use.

We have developed a novel superhydrophilic FPAR membrane with high tensile strength by in situ photocopolymerization and microphase separation, which can effectively separate oil-in-water emulsions with high separation efficiency and flux.

Today, the ever-growing serious environmental pollution caused by oil-contaminated water from the daily life of people as well as from industries demands the search for novel materials and strategies to realize oil/water separation with high efficiency.^1–5 Traditional separation technologies such as gravity separation, centrifugation, skimming, sedimentation, and flotation are useful for most of the separation processes. Unfortunately, low separation efficiency, high energy consumption and complex equipment have restricted the application of these technologies to some extent.^6–8 Other than that, it may be very difficult for them to separate emulsified oil/water solutions.⁹ Therefore, desirable materials for effective separation of oil/water emulsions are urgently needed. As a result, filtration polymer membranes have been considered to be a suitable technology for separating various emulsions, but suffer from low flux, surface fouling and poor mechanical properties.^2,9Recently, significant interests have been attracted to the design and preparation of oil/water separation membranes with special wettability by a combination of rough structure and surface chemistry.^2,10–14 Typically, these polymer membranes may be classed into two types, polymer coated mesh membranes and polymer porous membranes.^3,15–22 For polymer coated mesh membrane, it requires a mesh as a support which is capable of improving mechanical properties and rendering a micro-scale porous structure.² For example, Tuteja and co-workers developed a superhydrophobic mesh membrane coated with a blend of cross-linked poly(ethylene glycol)diacrylate and fluorodecyl polyhedral oligomeric silsesquioxane, which was valuable for separation of oil/water emulsions with droplet sizes larger than 1 μm.²¹ PVDF has been acknowledged as one of the main materials for manufacturing polymer porous membranes for separation of oil/water emulsions through a phase-inversion process.^1,2 In 2014, a superhydrophilic and underwater superoleophobic poly-(acrylic acid)-grafted PVDF (PAA-g-PVDF) membrane was fabricated by a salt-induced phase-inversion approach and applied to oil-in-water emulsions, however, the tensile strength of this membrane was not more than 0.64 MPa, which limited their practical applications.²²Polyarylate, a family of high-performance polymers, noted for their strength, toughness, chemical resistance, and high melting points.^23–26 Recently, Livingston and co-workers have demonstrated the formation of crosslinked polyarylate microporous membranes which have great potential for applications in molecular separations.²⁷ In previous studies, our group developed a simple procedure to fabricate a superhydrophobic and superoleophilic porous polyarylate membrane which could effectively separate oil/water mixtures.²⁸ In this communication, we reported the fabrication of a novel superhydrophilic sodium acrylate modified fluorinated polyarylate (SFPAR) membrane for efficient separation of oil-in-water emulsion by a combination of in situ photocopolymerization and microphase separation. It was very exciting that the as-prepared SFPAR membrane exhibited prominent mechanical strength and outstanding water permeability. Furthermore, the membrane also displayed excellent underwater superoleophobicity, antifouling performance and recyclability for long-term use, which highlight its potential for practical applications. Fig. 1 shows the formation of a SFPAR membrane via in situ photocopolymerization for endowing with the hydrophilic property of FPAR (Scheme 1a–c), followed a microphase separation (Scheme 1d and e) for obtaining the SFPAR membrane with porous structure. The experiments are described in detail in the ESI.^† Here, in situ photocopolymerization was applied for getting hydrophilic FPAR, which have the following advantages: good dispersibility of the formed acrylate copolymer in the FPAR matrix, low reaction temperature, and shortening the preparation time of membrane. After a microphase separation and a drying process, a white membrane was obtained by peeling from a substrate (Scheme 1f).Open in a separate windowFig. 1Water contact angle of the FPAR membranes as function of sodium acrylate mass fraction (a), water contact angle of the SFPAR and FPAR membranes as function of time (the insets are photographs of water drops on the membrane surfaces) (b), underwater–oil contact angle (c and d) and dynamic underwater–oil-adhesion of the SFPAR membrane (e and f). The underwater–oil contact angle were measured with 4 μL hexadecane droplet.Open in a separate windowScheme 1Schematic of the formation of a SFPAR membrane via in situ photocopolymerization and a limited micro-phase separation. The fluorinated polyarylate (FPAR) was fabricated by interfacial polymerization of bisphenol AF, terephthaloyl chloride, and isophthaloyl chloride (Fig. S1a^†). The number-average molecular weight of the obtained PAR is 93 000 and the polydispersity index is 1.76 (Fig. S2^†). The experiments are described in detail in the ESI.^†One of the main purposes of in situ photocopolymerization is to improve the wettability of FPAR by introducing carboxylate salts (Fig. S1b^†). Fig. 1a shows water contact angle of the FPAR membranes as function of sodium acrylate mass fraction. The results indicated that the value of water contact angle on the FPAR surface significant decreased with the increase of the sodium acrylate content. After the sodium acrylate content exceeded ∼9.5 wt%, the contact angle tended to equilibrium, less than ∼1°, which formed a superhydrophilic modified FPAR membrane (SFPAR). To further examine the wettability of water on the FPAR membranes, the water contact angles of the FPAR and SFPAR membrane as function of time was also measured (Fig. 1b). The pure FPAR membrane had the initial water contact angel of approximately 96.2° and the value of water contact angel almost kept stable after 100 s, exhibiting good hydrophobicity. On the contrary, the approach to introducing carboxylate to FPAR caused a significant differences. The SFPAR membrane had the initial water contact angel of approximately 40°. Interestingly, the value of water contact angel of the SFPAR membrane rapidly decreased to ∼1° in less than 7 s, illustrating outstanding superhydrophilicity, which is caused by the introduction of carboxylate sodium and the porous structure of the SFPAR membrane. Furthermore, the underwater–oil contact angle (OCA) and dynamic underwater–oil-adhesion of the SPAR membrane were also studied (Fig. 1c and d). An oil droplet was lifted up and contacted the SPAR membrane surface under water (Fig. 1c and d). It was observed that the oil droplet remained spherical and the underwater–OCA of this membrane is ∼161.7°, demonstrating excellent underwater superoleophobicity. From Fig. 1e to Fig. 1f, the oil droplet was forced to adequately contact the membrance surface and then moved to the left. During the moving process, the spherical oil droplet had no obvious deformation, also showing that the SPAR membrane had excellent antiadhesion to oil.ATR-FTIR spectra of the SFPAR and FPAR membranes are shown in Fig. 2a. For ATR-FTIR spectrum of the SFPAR membrane, besides the corresponding absorption peak of FPAR, The characteristic stretching peaks were obviously shown at 2850–3000 cm⁻¹ and 1457 cm⁻¹, respectively, resulting from –CH₂– and –CH₃, and –O–CH₂– groups of the crosslinked acrylate copolymer prepared by in situ photocopolymerization. The peak at 1569 cm⁻¹ was the asymmetric CO²⁻ (salts) stretching vibration in –CO₂Na of the formed acrylate copolymer. Moreover, the peak at 1640 cm⁻¹, which was attributed to the stretching vibration of vinyl bond, was not observed from the ATR-FTIR spectrum of the SFPAR membrane, indicating that the monomers were polymerized.Open in a separate windowFig. 2ATR-FTTR spectra of the SFPAR and FPAR membranes (a) and the overall XPS spectra of the SFPAR and FPAR membranes (b).XPS was performed to examine the surface chemical composition. Fig. 2b exhibits the overall XPS spectra of the SFPAR and FPAR membranes. There were three signals on the surface of the FPAR membrane attributed to C, O and F element whose atomic percentage was approximately 66.0, 20.6, and 13.4%, respectively. In comparison with the XPS spectrum of FPAR, the new signal appearing in the spectrum of the SFPAR membrane was attributed to Na element. The percentage of Na was estimated to be approximately 4.5 wt%, higher than the bulk content (2.3 wt%), and the F content of SFPAR membrane was obvious decreased and the O content is increased after in situ photocopolymerization, indicating the obvious surface enrichment of sodium carboxylate groups in the SFPAR membrane. Fig. 3 displays SEM images of the surface and cross section of the FPAR and SFPAR membranes. Apparently, the morphologies of the SFPAR membrane are different from those of the FPAR which can be attributed to the thermodynamics instability and the non-solvent Induce phase separation. The FPAR surface is smooth (Fig. 3a), while the SFPAP surface is porous and has a great number of micro nano-scale pores (Fig. 3c) and the pore size and pore distribution were calculated by Nano Measurer 1.2 (Fig. S3a^†). For the cross of the FPAR and SFPAR membranes, the former is dense and few pores can be found (Fig. 3b). However, the latter is loose and possesses many inter-connected nano-scale channels with a diameter of 50–200 nm (Fig. 3d). Different from the reported superhydrophilic membranes made from semicrystalline PVDF,^22,29,30 the FPAR is amorphous. According to the XPS and SEM results above, the possible formation mechanism of SFPAR membranes with porous structure prepared by in situ photocopolymerization and phase separation can be described as follows. As can be seen from Scheme 1, the FPAR, the monomers (BA, SA and TEGDA) and photoinitiator are first dissolved in THF to form the homogenous viscous solution (Scheme 1a and b). After in situ photocopolymerization of BA, SA and TEGDA occurs at room temperature, the crosslinked polyacrylate containing sodium carboxylate groups come into being in the viscous solution (Scheme 1c). During the immersion process (Scheme 1d), with the extraction of THF by the coagulation bath, the blend matrix of the amorphous FPAR and the crosslinked polyacrylate will gradually shrink and solidify. Simultaneously, a microphase separation occurs in the blend matrix due to the crosslinking of polyacrylate and the thermodynamics instability. Furthermore, sodium carboxylate groups attached to the polyacrylate network can absorb enough water in the blend matrix, ultimately leading to the formation of the wet membrane containing water. During the drying process, water is evaporated from the wet membrane and the porous structure appears in the membrane because the solidification of FPAR matrix restricts the movement of the polyacrylate segments. Finally, the SFPAR membrane with porous structure is obtained after the blend matrix is fully dried at room temperature (Scheme 1e and f). The hydrophilic sodium carboxylate groups will enrich in the SFPAR membrane surface and the inner surface of the micro nano channel due to the driving forces of surface free energy and hydrophilicity/hydrophobicity interactions (Fig. S3b, ESI^†), which makes it possible for the preparation of oil–water separation membrane.Open in a separate windowFig. 3SEM images of the FPAR and SFPAR membranes: the surface (a) and cross section (b) of the FPAR membrane; the surface (c) and cross section (d) of the SFPAR membrane. The inset is high-magnification SEM image of the SFPAR membrane surface.In this work, oil–water separation of the SFPAR membrane was carried out with a vacuum driven filtration system at 0.07 MPa. Toluene-in-water emulsion was employed to evaluate the separation ability of the membrane and the droplet size distribution of the emulsion is in the range from ∼900 nm to ∼8 μm (Fig. S4, ESI^†). Fig. 4a illustrates a self-made separation device and the separation result of toluene-in-water emulsion (the separation experiments are described in detail in the ESI^†). Compared with the milky white feed emulsion (up), the filtrate (down) is colorless from the appearance. A noticable difference was observed between the feed and the filtrate by the optical microscopy images. There appear a great many droplets in the image of the feed before filtration, however, no droplet can be viewed for the filtrate. Furthermore, the characteristic peak of toluene for the filtrate is not observed from UV-VIS spectrometer (TU-1901, Beijing Purkinje General Instrument Co., Ltd, China) in comparison with the feed (Fig. S5, ESI^†), and the oil content in the filtrate is 54 ± 17 ppm measured by a total organic carbon analyzer, indicating that the as-prepared membrane can successfully separate oil/water emulsion with high efficiency. The other two emulsions also have good separation efficiency (Table S1, ESI^†).Open in a separate windowFig. 4The vacuum driven filtration system and separation results for toluene-in-water emulsion (a) and change of the flux and flux recovery in the separation of a toluene-in-water emulsion over five cycles (b).Taking advantage of the reported method,^22,29 the flux and the antifouling property of the membrane were measured by the vacuum driven filtration system. Continuous separation of the toluene-in-water emulsion lasts for approximately 30 hours over five cycles and the flux is detected every an hour and six points were taken down within each cycle. The SFPAR membrane is gently washed by using DI water to dispose of surface adsorbent. As shown in Fig. 4b, the flux has a slight decline from ∼3800 to ∼3600 L m⁻² h⁻¹ within one cycle. Nevertheless, the membrane can recover fully to the initial flux after it is washed by water. The results show that the SFPAR membrane possesses a high flux and an outstanding antifouling performance for long-term use. Further studies will focus on the regulation of the pore size of the SFPAR membrane and get the most proper selectivity and penetration. Moreover, as one of the important factors in practical application, the tensile strength of the membrane was also tested by a testing machine (Fig. S6, ESI^†). Due to the porous structure, the tensile strength of the SFPAR membrane is ∼6.02 MPa, less than that of the FPAR membrane (∼27.59 MPa). However, the SFPAR membrane still has high mechanical property compared with the reported hydrophilic modified PVDF oil/water separation membrane.^14,17In conclusion, we have developed a novel superhydrophilic modified FPAR membrane with porous structure by in situ photocopolymerization of acrylate monomers and subsequent microphase separation. The results of ATR-FTIR and XPS demonstrated that sodium carboxylate groups was immobilized in the FPAR membrane by in situ photocopolymerization. When the sodium acrylate content was beyond ∼9.5 wt%, the as-prepared SFPAR membrane exhibited prominent superhydrophilicity, underwater superoleophobicity, and water permeability. The SFPAR membrane could effectively separate oil-in-water emulsions with high separation efficiency and high flux. Significantly, the obtained membrane possessed a good antifouling property and could be recycled for long-time use. From a practical perspective, the SFPAR membrane had a higher mechanical strength than traditional hydrophilic polymeric membranes with similar permeation properties. Therefore, we anticipate that our membrane will have high potential in practical application for treating wastewater from the daily life and industries.