Establishment of Master Cell Stock and Working Cell Bank of MDCK Lines and Selection and Evaluation of the Lines as Candidate Viral Substrates for Approval Production of Combinational Canine Attenuated-live Virus Vaccines

Under the prerequisite that the incidence of cancer or tumor in negative-control nude mice inoculated subcutaneously with primary feline or canine kidney cell cultures purified in vitro at passage 3 was 0 (0/22) and 0 (0/10), respectively. The incidence of the progressively-growing malignant tumor(MT) in positive-control nude mice inoculated subcutaneously with Hela cell cultures of KB, X, or NM20/X strain was 10/ 10, 25/25 and 5/51, respectively. The results showed that the incidence of tumor in nude mice with di-and hy-perploid YB strain of MDCK cell during 17 - 23 passages, with hyper- and hypoploid KA strain of MDCK cell during 6-8 passages, with hypoploid WB strain of MDCK cell on passage 6, with hyper-and hypoploid H strain of MDCK cell during 8-24 passages was 2/24, 6/10, 5/10 and 10/15, respectively. The chromosomal analysis results showed that the ratio of difference in the rate of modal chromosome number between high(mcs + n) and lowest (mcs)passages was not more than 5 - 15% and the structure aberrations was generally 0 -3 %. These results proved that the genetic characteristics of chromosomal number of cell lines determines their tumorigenicity, but it is species-specific. MDCK line has tumorigenicity no matter what its chromosome kary-otype is, at least it has very low tumorigenicity even when its modal chromosome number is hypoploid. It is thus evident that MDCK cell of WB or H strain can be approved as substrate for the preparation of attenuated viral vaccines, but MDCK cell of YB or KA strain can not be approved as substrate for the preparation of attenuated viral vaccines.

Dental pulp cell bank as a possible future source of individual hepatocytes

Mesenchymal stem cells(MSCs) as a source for regenerative medicine are now the subject of much clinical attention. There are high expectations due to their safety, low tumorigenic risk, and low ethical concerns. MSC therapy has been approved for acute graft-versus host diseases since 2015. Tooth-derived MSCs are known to have a great potential in their proliferation and differentiation capacities, even when compared with bone-marrow-derived MSCs. In particular, stem cells from human exfoliated deciduous teeth(SHEDs) are the best candidates for personal cell banking(dental pulp cell bank), because they can be obtained less invasively in the natural process of individual growth. SHEDs are known to differentiate into hepatocytes. There have been several studies showing the effectiveness of SHEDs on the treatment of liver failure in animal models. They may exert their effects either by repopulation of cells in injured liver or by paracrine mechanisms due to their immuneregulatory functions. Moreover, it may be possible to use each individuals' dental pulp cells as a future source of tailor-made differentiated hepatocytes in the context of a bioartificial liver or liver-on-a-chip to screen for drug toxicity.

Banking of perinatal mesenchymal stem/stromal cells for stem cellbased personalized medicine over lifetime:Matters arising

Mesenchymal stromal/stem cells(MSCs)are currently applied in regenerative medicine and tissue engineering.Numerous clinical studies have indicated that MSCs from different tissue sources can provide therapeutic benefits for patients.MSCs derived from either human adult or perinatal tissues have their own unique advantages in their medical practices.Usually,clinical studies are conducted by using of cultured MSCs after thawing or short-term cryopreserved-then-thawed MSCs prior to administration for the treatment of a wide range of diseases and medical disorders.Currently,cryogenically banking perinatal MSCs for potential personalized medicine for later use in lifetime has raised growing interest in China as well as in many other countries.Meanwhile,this has led to questions regarding the availability,stability,consistency,multipotency,and therapeutic efficiency of the potential perinatal MSC-derived therapeutic products after longterm cryostorage.This opinion review does not minimize any therapeutic benefit of perinatal MSCs in many diseases after short-term cryopreservation.This article mainly describes what is known about banking perinatal MSCs in China and,importantly,it is to recognize the limitation and uncertainty of the perinatal MSCs stored in cryobanks for stem cell medical treatments in whole life.This article also provides several recommendations for banking of perinatal MSCs for potentially future personalized medicine,albeit it is impossible to anticipate whether the donor will benefit from banked MSCs during her/his lifetime.

Allogenic banking of dental pulp stem cells for innovative therapeutics

Medical research in regenerative medicine and cellbased therapy has brought encouraging perspectives for the use of stem cells in clinical trials. Multiple types of stem cells, from progenitors to pluripotent stem cells, have been investigated. Among these, dental pulp stem cells(DPSCs) are mesenchymal multipotent cells coming from the dental pulp, which is the soft tissue within teeth. They represent an interesting adult stem cell source because they are recovered in large amount in dental pulps with non-invasive techniques compared to other adult stem cell sources. DPSCs can be obtained from discarded teeth, especially wisdom teeth extracted for orthodontic reasons. To shift from promising preclinical results to therapeutic applications to human, DPSCs must be prepared in clinical grade lots and transformed into advanced therapy medicinal products(ATMP). As the production of patient-specific stem cells is costly and time-consuming, allogenic biobanking of clinical grade human leukocyte antigen(HLA)-typed DPSC lines provides efficient innovative therapeutic products. DPSC biobanks represent industrial and therapeutic innovations by using discarded biological tissues(dental pulps) as a source of mesenchymal stem cells to produce and store, in good manufacturing practice(GMP) conditions, DPSC therapeutic batches. In this review, we discuss about the challenges to transfer biological samples from a donor to HLA-typed DPSC therapeutic lots, following regulations, GMP guidelines and ethical principles. We also present some clinical applications, for which there is no efficient therapeutics so far, but that DPSCs-based ATMP could potentially treat.

B细胞衔接蛋白BANK在小鼠实验性自身免疫性脑脊髓炎中的作用

目的:探讨B细胞特异性衔接蛋白BANK(B cell adoptor protein with ankyrin repeats)在小鼠实验性自身免疫性脑脊髓炎(Experimental autoimmune encephalomyelitis,EAE)中的作用。方法:MOG35-55多肽免疫C57BL/6鼠和BANK缺陷(BANK-deficient,BANK-/-)鼠制备EAE模型,观察实验动物临床症状及中枢神经系统的病理学变化;提取小鼠脑组织及脾脏,经流式细胞术检测中枢神经系统及外周免疫器官中的CD4+T细胞、CD8+T细胞及调节性T细胞的变化。结果:BANK-/-鼠EAE临床症状评分明显高于C57BL/6鼠,且体重减轻明显(P<0.05);HE染色结果显示,BANK-/-鼠较C57BL/6鼠炎症感染灶明显增多。流式细胞术结果显示,相对于C57BL/6鼠BANK-/-鼠中枢神经系统中CD8+T细胞百分比明显增多,而脾脏中调节性T细胞百分比明显减少,CD4/CD8比值倒置(P<0.05)。结论:B细胞衔接蛋白BANK的表达抑制EAE炎症反应。

Induced pluripotent stem cells, a giant leap for mankind therapeutic applications

Induced pluripotent stem cells(iPSC)technology has propelled the field of stem cells biology,providing new cells to explore the molecular mechanisms of pluripotency,cancer biology and aging.A major advantage of human iPSC,compared to the pluripotent embryonic stem cells,is that they can be generated from virtually any embryonic or adult somatic cell type without destruction of human blastocysts.In addition,iPSC can be generated from somatic cells harvested from normal individuals or patients,and used as a cellular tool to unravel mechanisms of human development and to model diseases in a manner not possible before.Besides these fundamental aspects of human biology and physiology that are revealed using iPSC or iPSC-derived cells,these cells hold an immense potential for cell-based therapies,and for the discovery of new or personalized pharmacological treatments for many disorders.Here,we review some of the current challenges and concerns about iPSC technology.We introduce the potential held by iPSC for research and development of novel health-related applications.We briefly present the efforts made by the scientific and clinical communities to create the necessary guidelines and regulations to achieve the highest quality standards in the procedures for iPSC generation,characterization and long-term preservation.Finally,we present some of the audacious and pioneer clinical trials in progress with iPSC-derived cells.

Drug-Induced Hemolytic Anemia: A Fatal Complication Further Under-Recognized in Sickle Cell Disease

Drug-induced immune hemolytic anemia (DIIHA) is commonly attributed to cephalosporins. Ceftriaxone is the most frequently administered cephalosporin in patients with sickle cell disease. We present a pediatric patient with severe DIIHA (hemoglobin < 2 g/dl) who survived. Since DIIHA often goes undiagnosed until late in the course, vigilance of DIIHA minimizes unnecessary diagnostic tests and therapies. DIIHA likely remains under-recognized in all patient subpopulations due to its rarity and overlapping presentations with other conditions. Distinction between exacerbation of chronic hemolysis and new onset of acute hemolysis poses a unique challenge in patients with sickle cell disease. A thorough analysis is warranted to better identify factors within the pediatric sickle cell population that may increase the predisposition for DIIHA, particularly due to ceftriaxone.

Review: Umbilical Cord Stem Cells

The use of umbilical cord blood as an alternative to bone marrow as a source of haematopoietic stem cells for the treatment of certain diseases has been on an increase since the first transplantation of umbilical cord-derived stem cells in 1988. In this paper, 6 of the latest case studies about the use of such stem cells have been discussed and are used as evidence to confirm the potential use of the umbilical cord as a source of haematopoietic stem cells for transplantation as treatment to various diseases. The debate over whether umbilical cord blood should be stored and why public banking facilities are preferred over those that are private have also been discussed in this paper.

Regenerative medicine using dental pulp stem cells for liver diseases

Acute liver failure is a refractory disease and its pro-gnosis, if not treated using liver transplantation, is extremely poor. It is a good candidate for regenerative medicine, where stem cell-based therapies play a central role. Mesenchymal stem cells(MSCs) are known to differentiate into multiple cell lineages including hepatocytes. Autologous cell transplant without any foreign gene induction is feasible using MSCs, thereby avoiding possible risks of tumorigenesis and immune rejection. Dental pulp also contains an MSC population that differentiates into hepatocytes. A point worthy of special mention is that dental pulp can be obtained from deciduous teeth during childhood and can be subsequently harvested when necessary after deposition in a tooth bank. MSCs have not only a regenerative capacity but also act in an anti--inflammatory manner via paracrine mechanisms. Promising efficacies and difficulties with the use of MSC derived from teeth are summarized in this review.

人脐带间充质干细胞生物学特性及其在难治性疾病中的应用进展

人脐带间充质干细胞(HUCMSCs)是一种多能干细胞,具有免疫调节功能、旁分泌功能和多向分化潜能等生物学特性。在一定条件下,HUCMSCs经诱导可分化为多种细胞,也可再生成其他组织器官,在临床上具有广泛的应用前景。目前关于难治性疾病多采用药物治疗,很难使受损或功能障碍的细胞恢复正常。因此,深入研究HUCMSCs在移植物抗宿主病、系统性红斑狼疮、肝纤维化、心血管疾病、呼吸系统疾病等疾病中的治疗效果,可以为难治性疾病的治疗提供新思路。同时,HUCMSCs库的建设和发展可促进HUCMSCs技术的产业化和临床应用,从而为临床提供高质量的HUCMSCs注射液。

小尾寒羊耳组织成纤维细胞系的建立与生物学特性研究

Taking Small Tail Han Sheeps ear margin as the study materials, we succeeded in establishing a fibroblast cell bank which content of samples was 52 by the method of explant culture directly and cryopreservation technique. The biological analysis results showed that population doubling time (PDT) of cells was approximately 24 h, diploid account for 90%～96% in chromosome. Confirmed by isoenzyme analysis,this cell line had no cross-contamination.The test results of the bacterium, fungi, virus, mycoplasma were negative. The transfection rate was high, and the extro-gene can be effectively expressed. Every indexes of the cell bank met all the standard quality controls of ATCC. It had not only preserved the germplasm resources of this important Small Tail Han Sheep on the cell level but also provided valuable material for the research of genome, postgenome and somacloning. Meanwhile, the setting-up of this technological platform will offer technology and theory for storing the cell genetic resources of other animals and poultry.

人角质形成细胞库的建立

目的 探索人皮肤表皮细胞分离、培养、传代、冻存及复苏技术。方法 采用细胞培养技术及免疫组化染色鉴定技术。结果 采用细胞培养技术体外分离培养了小儿包皮表皮细胞 ,培养的表皮角质形成细胞 ,经冻存、复苏后 ,此角质形成细胞仍既能增殖 ,又能分化 ,生长状态与新鲜分离的角质形成细胞相类似。

人骨髓间质干细胞库的初步建立

目的探讨建立生物学性状稳定的人骨髓间质干细胞(humanmarrowmesenchymalstemcells,hMSCs)库的可行性,为组织工程种子细胞的来源作初步探索。方法对分离得到的hMSCs采用液氮低温冻存,并在一定时间复苏培养,以流式细胞仪检测其表面抗原,在诱导培养基中对其行成骨和成软骨诱导培养,光镜、电镜观察细胞的形态,采用组织化学、免疫组织化学及分子生物学的方法检测成骨和成软骨诱导的特异标志物:碱性磷酸酶、型胶原、型胶原及骨钙素,研究其生物学性状。结果从骨髓中分离得到了纯度达96%以上的hMSCs,复苏后的hMSCs形态和表面抗原保持不变,可继续扩增10代以上,倍增时间40h。复苏后第2、6、10代hMSCs均保持较强的向软骨细胞、成骨细胞的分化能力。结论初步建立了hMSCs库,可为骨及软骨组织工程提供较好的种子细胞。

太行黑山羊成纤维细胞系建立与生物学特性研究

以太行黑山羊耳缘组织为材料,采用组织块直接培养法和细胞冷冻技术构建了成纤维细胞系,并进行了细胞活力测定生长动力学观察、微生物污染间检测、染色体标本制备、同功酶分析及生物学研究。结果表明:细胞群体倍增时间(PDT)约为24 h;细胞染色体中二倍体(2n=60)占主体,镜检细胞的百分比为98.2%;乳酸脱氢酶(LDH)、苹果酸(MDH)脱氢酶同工酶分析,没有其他物种污染;细菌、真菌、病毒、支原体检测阴性。该细胞库的各项指标均达到ATCC细胞系鉴定标准。此项研究不仅在细胞水平上保存了这一重要畜禽品种的种质资源,而且亦为其基因组和后基因组及体细胞克隆等研究提供宝贵实验材料。同时,此技术平台的建立必将为其他畜禽遗传资源细胞水平的保存提供技术与理论支撑和保障。

非洲绿猴肾细胞(Vero)无血清细胞库的建立

目的建立Vero细胞无血清细胞库。方法采用序贯适应和直接适应方法,将Vero细胞从有血清培养适应到无血清培养,建立Vero细胞无血清细胞主库和工作库,采用STR图谱并结合染色体计数对Vero细胞进行鉴别,并对Vero细胞无血清细胞库进行致瘤性和病毒外源因子检定。结果两种方法均能使Vero细胞适应到无血清培养,其中直接适应法可大大缩短适应代次,因此选择其作为建立Vero细胞无血清细胞库的方法。Vero细胞无血清主库和工作库的STR图谱与日本细胞库公布的Vero细胞STR图谱完全一致,Vero细胞库无外源因子污染,无致瘤性。结论Vero细胞无血清细胞库可用作生物制品生产用候选细胞基质。

海南省黎族永生细胞库的建立

采用ＥＢ病毒转化外周血Ｂ淋巴细胞同时加环孢菌素Ａ法，建立了海南黎族永生细胞库。该库共含有５４株“润黎”永生细胞株，供血者均身体健康，且三代无与不同民族或不同支系通婚史。其中男性占５６％，女性占４４％，有亲缘关系者仅占１３％

频危畜禽品种细胞库的构建与鉴定

我国是世界上畜禽种质资源最丰富的国家之一。近些年由于受诸多不良因素的影响而导致很多畜禽品种受到一定程度的威胁。笔者认为,通过构建濒危畜禽品种细胞库的途径以保存其种质资源,是目前最为理想的方法之一。因此,本文就构建濒危畜禽品种细胞库的细胞生物学发展概况、建库方法和技术以及鉴定等问题进行了论述,试图起到抛砖引玉的作用,并渴望通过同仁的共同努力,实现珍贵畜禽种质资源的长期保存。

MDCK细胞库的建立及其生物学特性研究

分别从美国典型培养物保藏中心(ATCC)、中国典型培养物保藏中心(CCTCC)、北京协和细胞资源中心及某企业引进4株MDCK细胞系,扩大培养后液氮冻存,分别建立种子细胞库和工作细胞库。细胞复苏后分别对每株细胞进行了活力、形态、生长曲线、微生物污染、核型及荧光蛋白质粒转染表达等特性研究。结果表明,4个来源的MDCK细胞均呈上皮型,生长状态良好;生长曲线呈S型;细菌、真菌、支原体检测都为阴性;染色体2n=78;外源质粒在该细胞中能进行复制和表达。建立的细胞库为开展MDCK细胞及流感细胞基质疫苗的研究提供了基础理论依据和细胞资源。

抗8种主要呼吸道病毒单抗杂交瘤细胞库的建立和临床应用

目的：为了对流感病毒甲型和乙型、副流感病毒１、２和３型、呼吸道合胞病毒及腺病毒３、７型主要呼吸道病毒进行快诊。方法：从１９８６到１９９７年应用杂交瘤单抗技术，制备出以上４类８种抗病毒单抗细胞库，连同用于连接物的抗碱性磷酸酶单抗细胞株，共有１１７株单抗细胞株。将以上４类８种病毒单抗连接抗碱性磷酸酶单抗复合物，以兔抗鼠搭桥进行ＡＰＡＡＰ桥联快速诊断。结果：获得满意结果，此技术结果清晰，无内源性酶干扰，只需１台普通光学显微镜，２～３ｈ可获得准确结果。本系列快速诊断试剂已获得国家４项新药证书及５项生产文号。结论：此抗主要呼吸道病毒单抗细胞库，不仅可提供快诊试剂，也可提供病毒亚型分析。

MDCK单克隆细胞库的建立及检定

目的:建立甲型H1N1流感病毒疫苗株高适应MDCK单克隆细胞库,用于培养生产流感病毒疫苗,为细胞代替鸡胚制备流感病毒疫苗提供保证。方法:用有限稀释法将MDCK细胞进行单克隆化,通过血凝和TCID50筛选甲型H1N1流感病毒疫苗株的高适应性单克隆化细胞株,扩大培养建立细胞库并进行检定。结果:共制备了97株单克隆化MDCK细胞,筛选到甲型H1N1流感病毒疫苗株高适应性MDCK单克隆细胞株,扩大培养建立了细胞库,经检定符合《中华人民共和国药典.三部》(2010版)对细胞库的要求。结论:建立了甲型H1N1流感病毒疫苗株高适应性MDCK单克隆细胞库,为细胞培养生产流感病毒疫苗奠定了基础。