Analysis of stem cell culture performance in a microcarrier bioreactor system koushik ponnuru1, jincheng wu1, preeti ashok1, emmanuel s. Algimatrix 3d culture system thermo fisher scientific us. A microcarrier is a support matrix allowing for the growth of adherent cells in bioreactors in 1967, microcarrier development began when van wezel found that microcarriers could support the growth of anchoragedependent cells. Us6048728a cell culture medium for enhanced cell growth. Animal cell culture media comprising peptides derived from rice us6692961b1 en 19961011. This facility further expands wuxis already extensive biologics manufacturing capabilities. When more cells are desired, the plates or flasks can be expanded by using more or larger vessels. Thelargest application is the use ofanima cells as vehicles in which togrow. The advantages of microcarrier technology in the vaccine industry include a ease of scaleup, b ability to precisely control. By day 10 of the culture, almost all microcarriers were populated, showing that hmscs can transfer to freshlyadded microcarriers as their surface area. It has been accepted for inclusion in cell culture engineering xv by an authorized administrator of eci digital archives. Harvest of human mesenchymal stem cells from microcarriers. At this time the stored cell culture medium was added back to the bioreactor for a final volume of 200 l. The company is based in berlin, germany, where it employs 60 people.
It will also be the largest biologics manufacturing facility of any kind in china. Microcarrier cell culture is typically carried out in spinner flasks, although other vessels such as rotating wall microgravity bioreactors or fluidized bed bioreactors can also support microcarrierbased cultures. These procedures are used routinely in our laboratories for the growth of vero cells on cytodex 1. To demonstrate cell binding on the 3d microcarriers, followed by cell culture, and highspeed imaging of cells on flowing microcarriers, we chose the mdamb231gfp breast cancer line. Shaped 3d microcarriers for adherent cell culture and. Microcarrierbased cell culture in the xdr singleuse stirred. Perform all activities under aseptic culture conditions. Incubate for 5 minutes, while examining the sponge under a microscope. Should optimize both ncsfbs and the 2me concentraction high glucose dmem 25 mm 15% ncs 75 ml ncs, or 15 tubes in 500 ml. Microcarriers are typically 125 250 micrometre spheres and their density allows them to be maintained in suspension with gentle stirring. Innovation cell automated simultaneous modeling of performance against provider prices tradeoff price vs performance seamless integration with csps and other commercial virtualization frontends single pane of glass for automation, orchestration and workload management across. As a result, specially treated beads have been optimized for cell culture and systems have been adapted for the routine production of large quantities of cells and viral vaccines giard, et al. Cell culture techniques have become vital to the study of animal cell structure, function, and differentiation and for the production of many important biological materials such as vaccines, enzymes, hormones, antibodies, interferons, and nucleic acids. Kinetics of rabies virus production by vero cells grown in batch culture mode in a 2 l bioreactor on 3 gl cytodex 1 and in vpsfm.
Microcarrier cell culture principles and methods 18114062 percoll methodology and applications 18111569 ficollpaque plus for in vitro isolation of lymphocytes 18115269 gst gene fusion system handbook 18115758 2d electrophoresis using immobilized ph gradients principles and methods 80642960 antibody purification handbook 18103746. The protocols described here can be modified for the growth of a variety of. Merck will use biochrom to strengthen its millipore process solutions business, which provides products and services to. Jun 27, 2017 although bare microcarriers were observed in the culture at day three by staining of nuclei, fresh microcarriers were added at this time point in anticipation of increasing cell mass. Microcarrier cell culture biorad laboratories, inc. Changing prices in the bio cmo market contract pharma. Cell culture medium can be added to the suspension if desired 0. Proteinfree cell culture media supplements are described consisting of synergistic combinations of medium components, which when added to cell culture media, either serum supplemented or serumfree, enhance cell growth, culture longevity and product expression. Discussion in verizon wireless forum started by g8rguy, mar 2, 2007. Recommended citation mark szczypka, scaling microcarrierbased cell expansion processes in cell culture engineering xv, robert kiss, genentech sarah. The 50 l culture was subsequently used to seed a 250 l culture by detaching the cells from the beads through trypsinization using tryple express enzyme, harvesting the detached cells through a 3 l harvestainer system, and adding the collected cells to the prepared 250 l s.
Many parameters can impact cell attachment and expansion on microcarriers including microcarrier surface, ph level, and medium. The microcarriers are sterile, readytouse, and available in closed systems packaging to be used directly with bioreactors. Cells grow as monolayers on the surface of the microcarriers, which are. Cisco has confirmed that it is endoflifing its universal small cell range.
The physical characteristics of solid beads limit the number of cell doublings that can be obtained in each culture step. Microcarriers can help to amplify cell numbers and maintain the appropriate phenotype for tissue repair and restoration of. For mammalian cell culture production, prices for smallscale projects have dropped, while prices for mediumscale projects have increased. Cell culture flasks are not feasible for industrial scale production when a thousandfold increase in.
Optimizing the hyperforma singleuse bioreactor for. In addition, the prices charged by cmos for ancillary. At 30 minutes posttrypsin introduction, cell detachment was satisfactory 90%. Corning microcarriers are designed to simplify your scaleup processes.
Additionally, the microcarriers are surface treated to enhance cell attachment, maximizing cell yield and viability. A variety of cell culture techniques are available, featuring the omni c3 cell culture bag. Enter your mobile number or email address below and well send you a link to download the free kindle app. Merck kgaa has struck a deal to buy german company biochrom, which makes cell culture media and buffer solutions, for an undisclosed price. All the necessary steps required for the cultivation of vero cells on microcarriers are described. Pdf a microcarrier cell culture process for propagating. L of prewarmed culture media to the tryple treated cell suspension and transfer to a well of a 48well plate containing approximately 350. Influence of culture conditions on vero cell propagation. Anchorage dependent mammalian cells are typically grown small scale in 2d culture vessels such as cell culture plates, cell culture flasks, or roller bottles, where they adhere to a specially treated growth surface 1. At the start, each bead has to be inoculated with 510 cells and cell growth terminates when cells make contact with each other, usually at. Past, present, and future of microcarrierbased tissue.
Mammalian cell culture october 2014 testing times by using the design of experiments approach in the production of chemicallyde. To begin discussion about any of these services, please contact us today by email or at. Then you can start reading kindle books on your smartphone, tablet, or computer no kindle device required. Traditional cell culture platforms are inadequate to perform highquality cell culture optimization experiments in highthroughput. Although bare microcarriers were observed in the culture at day three by staining of nuclei, fresh microcarriers were added at this time point in anticipation of increasing cell mass. A dynamic market for biopharmaceutical contract manufacturing services is seeing prices rise and fall. Incubate plate in a 37c incubator for 5 minutes or until cells have detached.
Influence of culture conditions on vero cell propagation on. Cell culture microcarriers cell expansion and yield corning. The availability of preadapted cells saves significant time and expense associated with the adaptation of cultures. Expression systems offers 293 and cho mammalian cell lines adapted to serumfree suspension culture in esf sfm. Microcarrier beads cell culture supplies sigmaaldrich.
Given the limitations of monolayer culture, such as expansion in a short period and maintenance of cell morphology, researchers have conducted indepth studies to improve the conditions. The top issue in tissue engineering is how to obtain more seed cells quickly and to preserve their characteristic morphology during in vitro expansion culture of cells. Microcarrier culture of hpscs also results in cell colonies that generally have less than 10 layers. Prices include the use of lampires standard medium. Microcarrier bead separation and cell harvest using thermo. The algimatrix 3d culture system is suitable for many cellbased screening and drug discovery procedures, including multicellular tumor spheroid assays mcts, hepatocyte and cardiomyocyte organogenesis studies, coculture studies, highthroughput drug screening, and embryonic stemcell 3d differentiation studies. Cultivation of mammalian cells using a singleuse pneumatic. Stirred microcarrier culture of hes cells br brazilian journal of medical and biological research 2009 42. It said, cisco announces the endofsale and endoflife dates for the cisco usc 3g small cell portfolio. Microcarrier based cell culture in the xdr singleuse stirredtank bioreactor figure 1. Monitor microcarrier cultures counting of adherent cells in.
With the aim of selecting appropriate culture conditions for the cultivation of vero cells at high cell densities, in this work the influence of several variables agitation rate, ratio of inoculated cells to microcarrier mass and fetal bovine serum concentration on cell growth on cytodex 1 microcarriers was studied. The selectline services enable small and largescale cell culture for established cell lines. Cell culture microcarriers cell expansion and yield. Ep0481791a3 culture medium for chocells and adapted cho. Animal cell culture media comprising nonanimal or plantderived nutrients us6103529a en 19961010. This article provides an overview on critical issues in cell culture of anchoragedependent cells and provides perspectives for future developments, in particular, with respect to the largescale amplification of anchoragedependent stem. Feb 05, 2015 from a cell culture device point of view, classically planar twodimensional culture systems have been used for the generation of cell mass. Using microcarriers in an xdr singleuse reactor makes largescale culture of these animal cells possible in a way that is efficient, practical, and economical. Certain limitedcharse cell culture microcarriers, inv. Microcarrier cell culture principles and methods 18114062 percoll methodology and applications 18111569 ficollpaque plus for in vitro isolation of lymphocytes 18115269 gst gene fusion system handbook 18115758 2d electrophoresis using immobilized ph gradients principles and methods 80642960. Merck kgaa buys cell culture company business chemistry world. The company posted a document on its website in april stating that usc products would be discontinued later this year, something that went unnoticed by the media. From the point of view of the cell production process, the combination of these features presents the following advantages. Microcarriers are able to support expansion and longterm selfrenewal of both hpscs and hmscs over multiple passages, proving the platforms capability to support production of clinically relevant cell numbers 80.
On one hand, inexpensive scaledown systems, such as well plates and shake flasks, cannot accurately reproduce operation in bioreactor systems. Wuxi to build worlds largest mammalian cell culture. Cell culture techniques have become vital to the study of animal cell structure, function and differentiation an for the production of many important biological materials such as vaccines, enzymes, hormones, antibodies, interferons and nucleic. Protocol expansion of cell line clones from single cells. Microcarrierbased cell culture in the xdr singleuse. When complete, this new facility will be the largest mammalian cell culture manufacturing facility using disposable bioreactors in the world. Additional services include roller bottle and bioreactor production. Changes in agitation rate, sparger choice, sparging rate, and impeller design can also impact cell attachment and subsequent expansion on microcarrier beads. Agitation was resumed at 50% the normal agitation rate and samples were removed every 5 minutes to assess the detachment progress. Microcarrier cell culture is typically carried out in spinner flasks, although other vessels such as rotating wall microgravity bioreactors or fluidized bed bioreactors can also support microcarrier based cultures.
In microcarrier culture, cells grow as monolayers on the surface of small spheres or as multilayers in the pores of macroporous structures that are usually suspended in culture medium by gentle stirring. Our mission is to offer our customers access to a robust catalog of worldleading brands to more effectively conduct cuttingedge research and development. Typically, the sponge begins to visibly degrade within 5 minutes after adding dissolving buffer. Scaling up cell and virus production can be challenging. Additionally, the microcarriers are surface treated to enhance cell attach. A microcarrier cell culture process for propagating rabies virus in vero cells grown in a stirred bioreactor under fully animal component free conditions. The protocols described here can be modified for the growth of a variety of anchoragedependent cell. Dec 02, 1980 as a result, specially treated beads have been optimized for cell culture and systems have been adapted for the routine production of large quantities of cells and viral vaccines giard, et al. This is the case when a cell population from a patient is expanded and used for grafting. Consistent, high density, final yields of more than 106 cellsml of culture medium are obtained. Cell culture techniques have become vital to the study of animal cell structure, function, and differentiation and for the production of many important biological materials such as vaccines. Mef plating density there is a wide range of mef plating densities used by researchers for mouse and human pluripotent stem cell culturing. Analysis of stem cell culture performance in a microcarrier.
An already existing application is skincellgrowth,while some\vhat morefuturistic examplesmay includegro\vth ofartificial pancreases andlivers. Stirred microcarrier culture of hes cells 515 issn 0100. Microcarriers offer a convenient method for growing adherent cells in bioreactors. On the other hand benchtop bioreactors, while accurate and scalable are. Analysis of stem cell culture performance in a microcarrier bioreactor system koushik ponnuru 1, jincheng wu 1, preeti ashok 1, emmanuel s. Microcarrier technology combines the necessary surface area, on which cells grow as a monolayer, with the benefits of a homogeneous suspension culture. Ep0481791a3 ep19910309596 ep99596a ep0481791a3 ep 0481791 a3 ep0481791 a3 ep 0481791a3 ep 19910309596 ep19910309596 ep 19910309596 ep 99596 a ep99596 a ep 99596a ep 0481791 a3 ep0481791 a3 ep 0481791a3 authority ep european patent office prior art keywords cells cho culture medium adapted adapted cho prior art date 199010. A profiles of cell density under sparged culture and surface. If no cell release is observed within 5 minutes, add another volume of. Microcarrier beads can be used in any application where anchoragedependent cells are to be cultured. Monitor microcarrier cultures counting of adherent cells. Microcarrier culture introduces new possibilities and, for the first time, allows practical highyield culture of anchoragedependent cells. Mef cell culture instructions all media and reagents used in the culture of this product should be warmed to 37c before use. Cell culture flasks are not feasible for industrial scale production when a thousandfold increase in production volume is needed.315 297 723 359 1371 770 440 314 474 1497 247 12 652 1066 1113 1402 512 1067 622 945 777 478 1173 425 208 1066 276 157 706 562 851 571 784 539 339 158 1138 1394 1300 1050 1418 237