动物细胞培养用多孔载体的研制

多孔载体是一种新型的用于动物细胞培养的优秀细胞支持物,其内部网状结构的小孔具有固定细胞和保护细胞免疫机损伤的功能,适合于贴壁细胞和悬浮细胞的培养,能提高培养密度,可应用于大规模培养系统。本文综述了多孔载体的物化性质、制作材料和制备方法。     

动物细胞高密度培养用多孔微球

动物细胞高密度培养用多孔微球王佃亮肖成祖（中国人民解放军航空医学研究所１０００３６）多孔微球（ｐｏｒｏｕｓｍｉｃｒｏｓｐｈｅｒｅｓ）又称多孔微载体（ｐｏｒｏｕｓｍｉｃｒｏｃａｒｉｅｒｓ）或大孔微载体（ｍａｃｒｏｐｏｒｏｕｓｍｉｃｒｏｃａｒｉｅｒｓ）,是近几年在国外发展起来的一项新的动物细胞高密度培养生物技术。它克服了固体微载体（ｓｏｌｉｄｍｉｃｒｏｃａｒ－ｉｅｒｓ）培养时细胞仅在表面生长、微...     

用多孔微载体Cytopore高密度培养CHO工程细胞生产rHEPO

在２．２Ｌｃｅｌｉｇｅｎ灌注培养系统中采用Ｃｙｔｏｐｏｒｅ多孔微载体培养产重组人促红细胞生成素（ｒＨＥＰＯ）的细胞Ｃ２。细胞密度达到１×１０７／ｍＬ,ｒＨＥＰＯ最高可达９．７ｍｇ／Ｌ。该载体具有表面积高,使用浓度低,便于扩大培养的特点,便于进行大规模培养     

自制多孔微球高密度培养Vero细胞的初步研究

多孔微球是动物细胞高密度培养的有效手段,它是1985年由Verax公司开创的,最初用于流化床生物反应器生产单克隆抗体,后来又出现了Percell和Siran系统系列多孔微球,并且使用的反应器种类和生产的产品都在增加,于是便成为一种新型的细胞培养手段而日益受到人们的重视。为此,我们在利用微载体进行Vero细胞高密度培养的同时,又对多孔微球的制备和培养工艺作了初步探索。     

两种微载体培养鼠肝细胞对低温保存后的影响

为了研究细胞培养方式对低温保存后细胞功能的影响,将大鼠肝细胞接种至两种不同微载体(实体微载体Cytodex、多孔微载体Cytopore),微重力高密度培养后于–80°C冻存(冻存速率是1°C/min,5%DMSO+0.4 mol/L山梨糖醇)。2周后复温细胞与载体材料,用显微镜观察细胞生长形态,计算细胞活力情况,并通过细胞代谢功能指标葡萄糖、白蛋白、尿素等的测定,反映细胞在两种载体培养和低温保存后的生长和代谢情况。结果表明,在细胞培养期间,Cytopore的MTT值、代谢指标值是Cytodex的1~1.5倍,低温保存后,Cytopore的各项指标与低温保存前差异不显著,而Cytodex的代谢指标差异显著。因此,大鼠肝细胞在微载体Cytopore的高密度培养及低温保存比Cytodex更有利于细胞的生长和代谢。     

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)×107／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67d连续换液培养中,细胞最高密度为2.64×107／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％～20％,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2100L上清中获得约80gu-PA(单链比例约为90％)。     

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)×107/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67d连续换液培养中,细胞最高密度为2.64×10⁷/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%～20%,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2100 L上清中获得约80guPA(单链比例约为90%)。     

用多孔微载体大规模培养rCHO细胞

多孔微载体是近年来发展起来的一种用于大规模高密度培养动物细胞的支持物,具有许多优点,如：容易固定细胞,适合于贴壁细胞和悬浮细胞的固定化连续灌流培养;细胞生长在载体内部,增加了细胞固定化的稳定性,可降低血清用量,适合长期培养;能保护细胞免受机械损伤,增加搅拌强度和通气量,强化反应器的传质;比表面积大,为细胞提供了充分的生长空间;细胞固定化过程简单无害,细胞能从长满细胞的微载体中自动转移到未长细胞的新载体中生长,接种方便,操作简单。特别适合于搅拌式、气升式、周定床和流化床等生物反应器的大规模培养〔1,2。尿激酶原(pro-UK)是一种重要的溶栓药物．与一般的生物医药制品相比,pro-UK给药量较大(约20mg／人～80mg／人),小规模生产不能满足市场需求。本文报道利用20L搅拌式反应器培养分泌pro-UK的重组CHO细胞的工艺条件,取得了初步结果。     

用多孔微载体大规模长期培养动物细胞的方法

长期大规模高密度动物细胞培养是生物制药产业中的关键技术,文中介绍了利用多孔微载体在中试规模生物反应器中长期大规模连续培养分泌尿激酶原的DNA重组中国仓鼠卵巢细胞（rCHO）的方法。     

用多孔微载体大规模长期培养动物细胞的方法

长期大规模高密度动物细胞培养是生物制药产业中的关键技术,文中介绍了利用多孔微载体在中试规模生物反应器中长期大规模连续培养分泌尿激酶 原的DNA重组中国仓鼠卵巢细胞（rCHO)的方法。     

Biomaterials aspects of porous microcarriers for animal cell culture.

Porous microcarriers are new support materials with important advantages in both industrial cell-culture processes and the culture of cells of medical importance. Porous microcarriers are now commercially available with internal architecture and surface chemistry suitable for culture of both anchorage-dependent and anchorage-independent animal cells.     

The inter-relationship between anchorage independence and tumorigenicity in early cultures of oral keratinocytes

This study examines the expression of anchorage independence and tumorigenicity in early cultures of oral rat keratinocytes. The epithelial cell lines originated from the palatal and the lingual mucosa of rats that had been painted with the carcinogen 4-nitroquinoline N-oxide. The colony forming efficiency (CFE) in gel culture of the cell lines derived from five squamous cell carcinomas of the tongue and palate predominantly increased with passage in culture. Carcinoma-derived cell lines that had a relatively high CFE (greater than 2.5%) formed tumours when transplanted to athymic mice, but cells in which the CFE was less than 2.5% were non-tumorigenic. Keratinocytes from a dysplastic palatal lesion were immortal, anchorage dependent and non-tumorigenic. A lingual papilloma cell line consistently expressed a very low CFE but was tumorigenic at the higher culture passages. The results show that the routine passage of cells in culture leads to the emergence of the anchorage independent and tumorigenic phenotypes in keratinocytes of malignant origin and, further, suggest that anchorage independence and tumorigenicity may exist as distinct phenotypes, with anchorage independence preceding tumorigenicity.     

Dynamic cell culture on porous biopolymer microcarriers in a spinner flask for bone tissue engineering: a feasibility study

Porous microspherical carriers have great promise for cell culture and tissue engineering. Dynamic cultures enable more uniform cell population and effective differentiation than static cultures. Here we applied dynamic spinner flask culture for the loading and multiplication of cells onto porous biopolymer microcarriers. The abilities of the microcarriers to populate cells and to induce osteogenic differentiation were examined and the feasibility of in vivo delivery of the constructs was addressed. Over time, the porous microcarriers enabled cell adhesion and expansion under proper dynamic culture conditions. Osteogenic markers were substantially expressed by the dynamic cell cultures. The cell-cultured microcarriers implanted in the mouse subcutaneous tissue for 4 weeks showed excellent tissue compatibility, with minimal inflammatory signs and significant induction of bone tissues. This first report on dynamic culture of porous biopolymer microcarriers providing an effective tool for bone tissue engineering.     

A new type porous carrier and its application to culture of suspension cells

A new type porous carrier was fabricated from a mixture of sodium alginate, bovine serum albumin and sodium bicarbonate. The porous space of the carrier is an assembly of void spaces. The carrier was successfully applied to the cultivation of suspension animal cells. In the culture, while both cells and carriers were held in suspension, the cells were entrapped hydrodynamically into the void spaces in the carriers. A culture of hybridoma cells using this carrier resulted in a cell density up to 5.7×10⁷ cells per ml-carrier.     

Recycle of Cytodex-3 in Vero cell culture

It is commonly considered not desirable to use microcarriers more than once in the cultivation of anchorage-dependent animal cells. However, our experiment contradicts this belief. The collagen-coated microcarriers, Cytodex-3, from a batch culture of Vero cells, were collected, cooled to 4, agitated in basic phosphate-buffered solution to detach the cells, and then fully washed to remove the cell debris. The microcarriers were then re-applied in cell culture. The rate of cell attachment, growth and metabolism on re-used carriers were found to be comparable to that of on new ones.     

动物细胞培养用生物反应器及相关技术

动物细胞大量培养是生产生物制品的重要途径,它用到的关键设备是生物反应器。根据培养细胞、培养载体、培养液混合方式的不同,生物反应器主要有搅拌式、气升式、中空纤维式、回转式等,其中搅拌式规模最大。回转式是NASA于20世纪90年代中期开发的一种新型生物反应器,被誉为空间生物反应器,可用于组织工程研究。与生物反应器配套的技术主要有灌注、微载体、多孔微球、转入抗凋亡基因等,可以有效地提高细胞密度,增加生物制品产量,提高质量。今后生物反应器研制主要朝两个方向发展:一是,以高密度培养动物细胞生产蛋白质药物为目的,二是以三维培养动物细胞(主要是人类细胞)再生组织或器官为目的。     

Hydrodynamic behaviour of animal cell microcarrier suspensions in split-cylinder airlift bioreactors

Hydrodynamic characteristics of suspensions of microcarriers used for culturing anchorage dependent animal cells are reported in split-cylinder internal-loop airlift bioreactors. Cell culture media are simulated using salt solutions that duplicate the ionic strengths of typical media. Effects of solids loading (0–30 kg·m^–3), microcarrier particle size (150–300×10^–6 m diameter) and density (1030–1050 kg·m^–3) on gas induced circulation of the slurry, mixing time, gas holdup and gas velocity requirements to attain complete suspension of solids are discussed for two reactors with aspect ratios of 7.6 and 14.5, but equal riser-to-downcomer cross-sectional area ratios of 1.0, aerated at low air flow rates (0–8×10^–6 m³·s^–1) through a sintered glass sparger with 110×10^–6 m diameter pores. The study covers the ranges of solids concentrations, types, densities, particle sizes and aeration rates that are of relevance in animal cell culture applications.Airlift bioreactors displayed suitable hydrodynamic characteristics for potentially supporting anchorage dependent cell cultures on microcarriers at carrier loadings similar to those that are currently used in stirred tank bioreactors. The minimum gas flow rates and the induced liquid circulation rates necessary to achieve and maintain suspension of the heaviest and the largest microcarriers were well within practicable limits, limits which have been shown to be withstood by animal cells in non-anchorage dependent suspension culture in airlift bioreactors. No floatation problems were encountered with the carriers, nor was sedimentation a problem so long as the identified minimum suspension criteria were met. Chisti's liquid circulation equation, originally intended for two-phase flow, applied to the three-phase gas-liquid-microcarrier systems.     

Cell number estimation via fluorescence measurement

Summary Spectrofluorometry was applied to estimate cell number in anchorage dependent cell culture. Ethidium bromide was employed to stain the DNA molecule. Good agreement of cell count was obtained between a Coulter counter and the peak fluorescent intensity of ethidium bromide intercalated within the DNA of lysed cells. This procedure can be utilized to estimate the number of cells growing on surfaces from which trypsinization is ineffective.     

Cell properties after repeated transplantation of spontaneously and of SV40 virus transformed mouse cell lines. I. Growth in culture.

"Spontaneously" or SV40 virus transformed AL/N mouse cell lines were passed repeatedly through syngeneic mice. Cell lines were re-established in culture from minced pieces of tumors in the presence of concentrated fetal calf serum or from tumor cells dispersed by trypsin. The aim of this study was to compare the two cell lines in regard to the selection processes which operate during such procedures by characterization of the resulting cell lines. Measurements of growth in tissue culture on substratum showed no significant difference between any of the transformed cell lines. The SV40 transformed cells and its derivative cells had a low anchorage requirement for growth. The greatest anchorage requirement for growth was in the normal untransformed cells and in the derivative cells from the "spontaneously" transformed cells which were established from minced tumors. The spontaneously transformed cells and all derivative cells had high tumorigenicity (TD50 is less than 10-2). The SV40 transformed cells had no observable tumorigenicity (TD50 is greater than 10-8), except when injected into irradiated mice (TD50 = 1-5 X 10-5 in the immunocompetent mice, 5 X 10-4 in the irradiated mice). The SV40 transformed derivative cells maintained their SV40 specific T antigen and their susceptibility to lysis by specific antiserum.     

Commitment to differentiation induced by retinoic acid in P19 embryonal carcinoma cells is cell cycle dependent

The rate at which P19 embryonal carcinoma cells in monolayer culture become anchorage dependent during differentiation induced by retinoic acid (RA) was investigated. In both nonsynchronized cultures and cultures synchronized by mitotic selection, the ability to grow in semisolid medium, characteristic of the malignant stem cell, decreased after a lag period of about 12 hr in the continuous presence of RA, prior to an increase in cell generation time. However, striking differences between synchronized and nonsynchronized cultures were observed in their commitment to differentiation following RA removal. After only 2 hr of exposure to RA, synchronized cells continued a program of differentiation in which they became anchorage dependent, while at least 24 hr of exposure was required for exponentially growing cells to become similarly committed. Induction of anchorage dependence by RA was also strikingly cell cycle dependent; 2 or 4 hr of exposure of synchronized cells to RA in G1 phase, when the intrinsic capacity for soft agar growth is low, was sufficient to commit cells to anchorage dependence, but a similar exposure in S phase was not. Together, these results suggested that interactions between cells in different cell cycle phases in asynchronous cultures influenced commitment since exposure to RA for more than one cycle (13 hr) was required for all cells to become anchorage dependent. Increased plasminogen activator secretion and epidermal growth factor binding, markers of certain differentiated cell types, increased only 3 and 5 days after RA addition, respectively, and were not induced by pulsed exposure to RA of less than 24 hr, even in synchronized cells.