Some biological characteristics of hybridomas and parental myelomas cultivated in serum-free medium for long-term period

It has been developed in this laboratory that a serum-free medium, designated asDMI,is adaptive for long-term culture,freezing and resurgence in liquid nitrogen,and cloningof hybridoma and parental myelomas as well as for cell fusions.In this report,it is describedthat the myeloma cells grown in DMI for more than 3 months maintained their biological charac-teristics such as induction of aseites and subcutaneous somatic tumor in BALB/c mice and toler-ance toward 8-azaguanine,ete..However,the ability of the tumor cells to attach to glass wallwas decreased.The selecting assay of these cells in HAT medium(medium containing hypoxan-thine,aminopterin and thymidine)showed that the death time in DMI was similar to that in theconventional 15％ newborn calf serum-supplemented medium(RPS15).The hybridoma cellsadapted in DMI secrete monoclonal antibodies in quantities comparable to those produced inRPS15.

Novel sericin-based hepatocyte serum-free medium and sericin's effect on hepatocyte transcriptome

AIM To develop a novel hepatocyte serum-free medium based on sericin,and to explore the effect of sericin on the hepatocyte transcriptome.METHODS A controlled trial comparing novel serum-free medium and other media: C3 A cells were cultured in our novel serum-free medium,Hepato ZYME,complete medium(DMEM/F12 with 100 m L/L FBS),and DMEM/F12,andthen cell attachment,proliferation,and function as well as the biocompatibility of the media were assessed.A comparative study of serum-free media with or without 2 mg/m L sericin: the effect of sericin on C3 A growth was assessed by cell viability and proliferation,the effect of sericin on C3 A cell cycle distribution was determined by flow cytometry,and the effect of sericin on the C3 A transcriptome was assessed by gene-chip array and RT-q PCR.RESULTS More C3 A cells attached to the plate containing our serum-free medium than to those containing Hepato ZYME and DMEM/F12 at 24 h post-seeding.Both the viability and proliferation rate of C3 A cells in sericin-based serum-free medium were superior to those of cells in Hepato ZYME and DMEM/F12(P < 0.001).The content of albumin and urea in our serum-free medium was significantly higher than that in Hepato ZYME and DMEM/F12 throughout the whole culture period(P < 0.001) and was similar to that in complete medium at day 3,4,and 5.In part 2,cell viability and proliferation were greater in the presence of 2 mg/m L sericin(P < 0.001),as was the proportion of cells in S phase(16.21% ± 0.98% vs 12.61% ± 0.90%,P < 0.01).Gene-chip array analysis indicated that the expression of CCR6,EGFR,and FOS were up-regulated by 2 mg/m L sericin,and RT-q PCR revealed that the expression of CCR6,EGFR,FOS,AKT1,JNK1,NFk B1,MMP-9,MEK2,ERK1/2 and MYC was upregulated by 2 mg/m L sericin(P < 0.05).CONCLUSION We developed a novel hepatocyte serum-free medium.Sericin probably enhances cell attachment through the CCR6-Akt-JNK-NF-κB pathway and promotes cell proliferation through CCR6-mediated activation of the ERK1/2-MAPK pathway.

Cholesterol requirement for growth of rodent parental myeloma cells in serum-free medium

Cholesterol,a major lipid component of plasma membrane,is thoughtto have profound effects on the structure and function of cells.Mostanimal tis-sues are capable of synthesizing cholesterol de novo from acetate;however,thereare relatively few mammalian cells in vitro expressing an absolute requirement foran exogenous source of cholesterol.In this paper,it was shown that IR983F(983)rat myeloma cells and P3X63-AgS-U1(P3U1)and P3X63-Ag8.653(653)mousemy eloma cells which had been cultivated in serum-free medium for more than 6months required cholesterol in vitro for growth in serum-free medium.Optimalgrowth of P3U1,653 and 983 occurred in cholesterol concentration of 5,10 and15μg/ml,respectively.Moreover,it was demonstrated that the cholesterol couldbe replaced by human low density lipoprotein in a concentration of 10μg/ml butnot by mevalonic acid lactone.In contrast to the parental myeloma cells,hybridoma cells derived from the mouse myeloma cells which had been cultivatedin serum-free medium for more than 6 months did not require cholesterol.

MDCK细胞无血清悬浮培养技术在流感疫苗研究与生产中的应用进展

流感是一种常见的呼吸道疾病,由流感病毒引起,接种疫苗是预防流感的有效手段。MDCK细胞(Madin-Darby Canine Kidney Cells)具有易于培养和高产量的特点,可以支持流感病毒的复制和增殖,被广泛用于流感疫苗的研究和生产。随着对流感病毒研究的不断深入,为了进一步拓展MDCK细胞用于流感病毒研究和工业应用的能力,研究人员开始发展MDCK细胞无血清全悬浮培养技术。通过长期的培养和优化,驯化的MDCK悬浮细胞株可以更好地适应流感病毒的生长环境,提高流感病毒的产量和感染性。总之,MDCK细胞无血清悬浮培养技术在流感病毒研究和工业应用中发挥着重要作用,为流感疫苗生产和抗流感药物研发提供更好的工具。同时也必须重视MDCK细胞的安全性,采取合适的措施来确保其应用的安全性和可靠性。

Pilot-Scale Production of Lyophilized Inactivated Rabies Vaccine Candidate in Vero Cells under Fully Animal Component-Free Conditions Using Microcarrier Technology and Laboratory Animal Trials

The upstream process was carried out in an animal component-free medium on Cytodex 1 microcarriers. Recombinant trypsin is a non-animal derived protease used as an alternative to animal-derived trypsin. To inactivate recombinant trypsin, a soybean trypsin inhibitor (STI) should be added to the medium. A protocol was first tested in T-flasks and then passaged to 500 mL and 3 L spinner flasks. Cell detachment was completed in 10 - 12 min, and 0.4 g/L STI was added to a 3L spinner, and cells were transferred into a 30 L stirred tank bioreactor. On day 5, the cell density had reached its maximum (around 1.8 × 106 cells/mL). At an MOI of 0.3 with serum-free medium conditions, cell infection yielded a maximal rabies virus titer of 1.82 × 107 FFU/mL at 5 days. All cell culture conditions and virus growth kinetics in serum-free media were investigated. In conclusion, Vero cells were grown on Cytodex 1 with serum-free media and a high amount of rabies virus was obtained. A mouse challenge was used to determine the immune response to an inactivated rabies virus vaccine candidate. Also, we evaluated inactive rabies vaccine candidate safety, and immunogenicity in mice, sheep, horses, and cattle. We found that no horses, sheep, or cattle who were given vaccine IM at 3.2 IU/dose exhibited any clinical sign of disease and all developed high VNA titers (up to 10.03 IU/mL) by 3 - 4 WPI. After the accelerated stability studies, the lyophilized inactivated rabies vaccine candidate showed enough antigenic potency (2.6 IU/mL) in the mouse challenge test. Also, 18-month long-term stability studies showed enough immune response (1.93 IU/mL) on day 14. The activity of the vaccine candidate showed a good immune response and safety criteria that meet WHO requirements. This is the first pilot-scale mammalian cell-based viral rabies vaccine production study in Türkiye that used microcarriers.

STUDY ON REPRODUCTIVE ENDOCRINOLOGY OF HUMAN PLACENTA--CULTURE OF HIGHLY PURIFIED CYTOTROPHOBLAST CELL IN SERUM-FREE HORMONE SUPPLEMENTED MEDIUM

A new method of long-term culture of cytotrophoblast cells in serum-free medium has been developed. Cytotrophoblast cells were isolated with cold trypsin and purified by unit gravity sedimentation through BSA density gradients. The cells were cultured in the FD medium with supplement of EGF, insulin, transferrin and sodium selenite. They could survive over three weeks. The results showed that both EGF and insulin stimulated hCG and progesterone secretion and that sodium selenite elevated hCG output but not progesterone secretion. Transferrin produced synergistic effect with EGF and insulin on hCG and progesterone secretion but it was ineffective when used alone. This study demonstrates that the four growth factors mentioned above are essential for the survival of cytotrophoblast cells in vitro. It is therefore suggested that EGF, insulin and selenium may possibly be involved in the regulation of hCG and progesterone secretion in the human placenta.

Enrichment of breast cancer stem cells using a keratinocyte serum-free medium

Background Keratinoyte serum-free medium （K-SFM） is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer stem cells （CSCs） using K-SFM. Methods＇ A K-SFM was used to enrich CSCs from two breast cancer cell lines and a primary culture of breast cancer. RPMI-1640 supplemented with 10% fetal calf serum （FCS） was used as a control. CSCs were identified with flow cytometry using CD44＋/CD24-as molecular markers. The expression of a variety of CSC markers （Oct-4, ABCG2, Nanog, N-cadherin, and E-cadherin） was analyzed with real-time PCR. Results Much higher percentage of CSCs was achieved with K-SFM： 17.3% for MCF-7 cells, 17.4% for SKBR-3, and 20.0% for primary breast cancer culture. Less than 1% CSC was achieved using RPMI-1640 supplemented with 10% FCS. In comparison to the CSCs obtained with RPMI-1640, CSCs in the K-SFM expressed higher levels of Oct-4, ABCG2, Nanog and N-cadherin, and lower level of E-cadherin. Conclusion K-SFM is an optimal culture medium to maintain and to enrich breast CSCs.

Application of Serum-Free Culture Medium for Preparation of A-NK Cells

To compare the differences between proliferation and cytotoxicity of adherent natural killer （A-NK） cells cultured with serum-free medium AIMV and standard serum-containing medium in vitro, and also observe the assisting effect of IL-12 on the activation and the morphology character of IL-2-treated A-NK cells, cellular proliferation was evaluated by MTT method in vitro. The morphology of the target cells killed by A-NK cells was observed through electroscope. All of the A-NK cells cultured in serum-free medium AIMV could rapidly proliferate and keep high cytotoxicity compared with that in standard serum-containing medium. A-NK cells activated by both moderate-dose IL-2 and IL-12 were superior to the high-dose IL-2-treated A-NK cells. These data indicated that serum-free medium AIMV could replace standard serum-containing medium for culturing A-NK cells, and moderate-dose IL-2 and IL-12 could reduce side effects caused by high-dose IL-2. The study provided a new experimental basis for experimental and clinical preparation of A-NK cells. Cellular ＆ Molecular Immunology.

脐带间充质干细胞无血清培养体系的筛选

目的:筛选培养脐带间充质干细胞的无血清培养基。方法:采集新生儿脐带标本,将脐带剪成组织碎块,随机分为A组、B组、C组,分别加入MesenCult、Ultraculture和AM-V 3种不同的无血清培养基进行培养。观察3组脐带原代细胞游出时间和细胞形态,检测细胞的增殖能力、分化能力和表面抗原的表达。结果:A组、B组、C组原代细胞从组织块长出的平均时间分别是17 d、6 d、7 d。显微镜下观察可见,从组织块游出的细胞散在分布,呈短梭形或多角形,形态饱满,折光性好,生长融合时A组和B组细胞呈旋涡状生长,细胞折光性好,大小均一,C组细胞细长,折光性稍差,无明显的旋涡状生长的特征。3组均表达CD105、CD90、CD73抗原,不表达CD34、CD45、HLA-DR抗原;3组各标志物的表达差异均无统计学意义(P>0.05)。3组间充质干细胞均具有向成脂细胞、成骨细胞分化的能力,C组分化能力弱于A组和B组。结论:B组培养体系用时较短,干细胞特性保持较好,价格适宜,可以作为大规模培养人间充质干细胞的首选培养基。

多孔微载体无血清培养rCHO细胞生产u-PA

在 30L搅拌式反应器中无血清培养分泌尿激酶型纤溶酶原激活剂 (u PA)的DNA重组CHO细胞 ,定期部分更换Cytopore多孔微载体 ,使生长在多孔微载体中的细胞不断更新繁殖 ,解决大规模细胞培养中的细胞凋亡问题。在 91d连接换液培养过程中 ,细胞密度可维持在 (1 3～ 2 6 )× 10 7/mL ,活细胞比率维持在 90 %以上。在7 5L搅拌罐中培养细胞 ,利用外部周期性压力振荡刺激并结合载体更新技术 ,可减轻密度效应对细胞生长和表达的影响 ,在一定程度上提高细胞在高密度培养条件下的表达水平。在 6 7d连续换液培养中 ,细胞最高密度为 2 6 4× 10 7/mL ,活细胞比率维持在 95 %以上。与稳压操作相比 ,利用周期变压刺激技术可提高产量 10 %～ 2 0 % ,且可降低葡萄糖厌氧代谢生成乳酸的转化率 ,利用 4步纯化工艺 ,从含u PA约 135g的 2 10 0L上清中获得约 80 gu PA(单链比例约为 90 % )。

蛋白水解物在动物细胞培养中的应用研究进展

蛋白水解物是蛋白质经水解得到的混合物,其主要成分为肽类。蛋白水解物可优化动物细胞培养基的组成,并且其中所含部分肽段可作为外部分子信号对细胞代谢、生物合成、生长、凋亡及产物表达等生命活动产生特殊的调控作用,因而被广泛应用于动物细胞培养领域,以生产单克隆抗体、疫苗、干扰素等生物制品。其作为无血清或低血清培养基的营养成分,可消除或降低血清带来的病毒微生物污染。随着蛋白水解物生产工艺的改进及优质产品的问世,其必将在细胞培养等生物技术领域发挥越来越重要的作用。

用无血清培养基在填充床生物反应器生产 rHuEPO

在填充床生物反应器用含５％ＦＢＳ的ＤＭＥＭ：Ｆ１２培养基培养产重组人促红细胞生成素（ｒＨｕＥＰＯ）的细胞Ｃ２８～１０ｄ后，使用自制的无血清生产培养基（ＳＦＭｐ）生产ｒＨｕＥＰＯ。ＳＦＭｐ培养基既能维持细胞生长，又能生产ＥＰＯ，也便于纯化分离ｒＨｕＥＰＯ。使用填充床生物反应器培养细胞，能维持培养２０～２５ｄ，ｒＨｕＥＰＯ表达水平达１２～２８４ｍｇ／Ｌ之间，反应器的产率达到７１０ｍｇ／Ｌ／ｄ，比滚瓶的产率增加１２～１４倍。葡萄糖最高消耗量达到２１ｇ／Ｌ／ｄ，细胞培养密度最高达到３０×１０７／ｍｌ以上，每次可收无血清培养上清８０～８７Ｌ。由于细胞被固定在聚酯片上，培养上清中脱落细胞很少。观察了反应器的乳酸和氨的含量，其结果表明乳酸和氨含量分别低于３５ｇ／Ｌ和５ｍｍｏｌ／Ｌ，不影响产物的表达。经过多批培养和生长ｒＨｕＥＰＯ的结果表明，自行配制的ＳＦＭｐ培养基在该反应器能有效地维持细胞生长和生产ｒＨｕＥＰＯ。

无血清培养的大肠癌Colo205细胞生成肿瘤干细胞球的研究

目的通过含生长因子的无血清培养基(SFM)培养大肠癌Colo205细胞,分离并扩增出肿瘤干细胞球。方法SFM培养大肠癌Colo205细胞,以含血清培养基(SSM)组为对照。观察细胞形态,检测正常肠道干细胞标志Musashi-1的表达,更换培养基为SSM诱导其分化,分别检测SFM组细胞分化前、分化后以及SSM组细胞表面标志CD133、CD44表达的改变,分析各细胞周期的比例,最后进行核型分析。结果Colo205细胞能在SFM中存活、增殖并形成肿瘤干细胞球,高表达Musashi-1,SFM组细胞分化前CD133、CD44的表达高于SSM组和SFM组分化后,差异有统计学意义(P<0.05),SFM组分化后与SSM组相比差异无统计学意义(P>0.05)。细胞周期分析发现肿瘤干细胞球处于高增殖状态,核型分析发现SFM组细胞与SSM组细胞染色体条数未见明显差别。结论含生长因子的SFM培养Colo205细胞,可生成肿瘤干细胞球,这种细胞球富集了肿瘤干细胞。

无血清培养基与完全培养基体外诱导扩增CIK细胞分泌细胞因子水平的比较

目的比较无血清培养基（AIMV）与完全培养基（CM）体外诱导扩增CIK细胞及CIK细胞分泌细胞因子水平。方法分别用AIMV及完全培养基加入4种细胞因子（IFN-γ、IL2、IL-1及OKT3）将脐带血单个核细胞（CBMNCs）诱导成CIK细胞，比较细胞的增殖能力、细胞表型及分泌细胞因子水平。结果与完全培养基比较，无血清培养基培养的CIK细胞增殖高峰较晚（14～17天），但增殖倍数高，在细胞表型、分泌细胞因子水平方面两种培养基培养的CIK细胞均无明显的差别（P〉0．05）。结论无血清培养基可代替完全培养基。

无血清培养基与完全培养基对体外诱导免疫细胞支持作用的比较

目的 :多方面比较无血清培养基AIMV与完全培养基对体外诱导免疫细胞支持作用。方法 :分别用AIMV及完全培养基在IFN γ ,IL 2 ,及抗 CD3单抗存在的条件下培养人外周血单个核细胞 ,比较细胞的增殖能力、细胞表型、及细胞因子分泌能力 ,并比较不同培养基培养细胞回输体内后的抑制病毒效果。结果 :与完全培养基培养细胞相比 ,无血清培养基AIMV培养细胞增殖能力与之相当 ;CD2 5的表达率增高 ,表达持续时间延长 ;细胞因子IFN γ的分泌时间延长 ;回输体内后的抑制病毒作用更明显。结论 :无血清培养基AIMV用于培养临床治疗用的免疫细胞 ,综合效果优于完全培养基。

人牙周膜成纤维细胞在无血清培养液中的生长特性

目的 :探讨人牙周膜成纤维细胞在无血清培养液中的生长特性。方法 :用倒置显微镜和MTT法观察人牙周膜成纤维细胞在无血清培养液中的生长和增殖变化。结果 :人牙周膜成纤维细胞在无血清培养液条件下可以生长和增殖 ,但与含血清培养液相比 ,其增殖速率变缓 ,分化明显。结论 :无血清培养液可应用于人牙周膜成纤维细胞的体外培养和实验研究中。

哺乳动物细胞无血清培养基研究进展

随着哺乳动物细胞培养种类、规模和应用范围的不断扩大以及安全性能要求的不断提高,无血清培养基的研制已经成为细胞工程领域的重要课题。许多类型的哺乳动物细胞无血清培养基配方得到了研究开发和优化,且有些已经进入第3代无蛋白无血清培养基研制阶段。论文重点概述了无血清培养基在添加因子作用机制、细胞分化条件、细胞和组织移植以及肿瘤治疗等方面的研究进展。

神经干细胞提取与培养方法的比较研究

目的探讨神经干细胞(neural stem cells,NSCs)提取、培养的最佳方法,为NSCs的基础研究提供技术参考。方法对NSCs的不同提取、培养方法进行比较:提取胎鼠与乳鼠的大脑皮质、采用不同类型培养基、不同接种密度、不同培养方法、不同换液方法、不同传代方法,观察NSCs的成球率和NSCs未分化状态的稳定性。用多功能微孔板读数仪检测NSCs活性。结果 (1)胎龄14 d胎鼠较新生24 h内乳鼠的大脑皮质提取NSCs的比例高,杂细胞少,形成的神经球数量多。(2)采用无血清培养基,NSCs可以稳定在未分化状态;而采用含血清培养基,NSCs多数分化为神经元和胶质细胞。(3)以1.0×109·L^(-1)密度接种的NSCs,培养形成的神经球数量多,且状态良好。(4)悬浮培养法较贴壁培养法,可以形成稳定的神经球,有利于保持NSCs处于未分化的状态。(5)半量换液法和只加液不弃液法培养形成的神经球的状态优于全量换液法。(6)原代和1代的神经球用机械吹打法传代后,NSCs的活细胞比例高,再次形成的神经球状态好,且较其他两种方法简便;2代及以后的神经球用Accutase酶传代后,NSCs的活细胞比例高,再次形成的神经球状态好。(7)14 d胎鼠Accutase酶消化组的NSCs活性高于另外3组,差异均具有显著性(P<0.05)。结论提取胎龄14 d胎鼠的大脑皮质,采用无血清培养基,以1.0×10~9·L^(-1)1的密度接种于25 cm^2培养瓶,采取悬浮培养法,每2~3 d加液1~1.5m L,每6~7 d传代1次,所得到的NSCs增殖分裂旺盛,成球率高,神经球状态良好,可以保持稳定的未分化状态。

无血清培养基与完全培养基对体外诱导扩增CIK细胞及抗肿瘤活性的比较研究

目的:比较无血清培养基AIMV与完全培养基对体外诱导扩增CIK细胞的效果。方法:分别用AIMV及完全培养基加入4种细胞因子(IFNγ、IL2、IL1及OKT3)将脐带血单个核细胞(CBMNCs)诱导成CIK细胞,比较细胞的增殖能力、细胞表型、对肿瘤细胞的增殖抑制作用及其诱导肿瘤细胞凋亡等几个方面。结果:与完全培养基比较,无血清培养基AIMV培养的CIK细胞增殖高峰较晚(14～17d),但增殖倍数高(倍),在细胞表型、对三株肺癌细胞的生长抑制作用及不同效靶比诱导细胞凋亡方面两种培养基培养的CIK细胞差异无统计学意义,P>0.05。结论:无血清培养基AIMV可代替完全培养基。

无血清专用培养基体外快速培养临床级树突状细胞的实验探讨

目的:探讨采用无血清专用培养基从肺癌患者外周血单核细胞快速培养树突状细胞(DCs)的方法。方法:将10例肺癌患者外周血单核细胞同时采用含人AB型血清培养基和无血清DCs专用培养基(DCMedium)培养,5例于第7天加入促成熟细胞因子组合,第9～10天收获成熟DCs,另5例于第5天加入促成熟细胞因子组合,且TNF-α剂量加大5倍,第7天收获成熟DCs。结果:DCMedium培养的DCs形态变化快,细胞出现突起的时间早;9～10天收获的两种培养基培养的DCs性能无差异;7天收获的DCs中,DCMedium培养的DCs得率和纯度、共刺激分子表达水平、刺激T细胞增殖能力均优于含血清培养基。结论:无血清DCs专用培养基可以在体外快速培养临床级DCs,为肿瘤免疫治疗奠定基础。