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1、Microcarrier Cell Culture Scale-Up Methods18-1020-61 Edition AAAntibody PurificationHandbook 18-1037-46The Recombinant Protein HandbookProtein Amplification and Simple Purification18-1142-75Protein PurificationHandbook 18-1132-29Ion Exchange ChromatographyPrinciples and Methods18-1114-21Affinity Chr

2、omatographyPrinciples and Methods18-1022-29Hydrophobic Interaction ChromatographyPrinciples and Methods18-1020-90Gel FiltrationPrinciples and Methods18-1022-18Handbooksfrom Amersham BiosciencesReversed Phase ChromatographyPrinciples and Methods18-1134-16Expanded Bed AdsorptionPrinciples and Methods1

3、8-1124-26Chromatofocusingwith Polybuffer and PBE50-01-022PBMicrocarrier cell culturePrinciples and Methods18-1140-62 1Microcarrier Cell CultureScale-Up MethodsContentsChapter 1Microcarrier cell culture. 3 Introduction . 3 Applications . 3 Cell attachment. 4 Requirements for an optimum microcarrier.6

4、 Properties and characteristics. 6 Instructions for use. 8 Preparation . 8 Inoculum . 8 Culture Procedure.8 Monitoring cell growth.8 Harvesting cells.9 Scaling up by serial subcultivation. 9 Equipment recommendations.10 Industrial applications.11 Trouble Shooting:.12Chapter 2An Analysis of Alternati

5、ves and Process Development.15 Introduction . 15 Process overview. 16 Provisions . 16 Process themes to be evaluated. 17 Principal process flow. 17 Equipment used. 17 Process outline and suggested experiments. 18 Process 1a.18 Process 1b.22 Process 2a.24 Process 2b.27References. 282Chapter 1Microcar

6、rier cell cultureIntroductionAnimal cell culture, vital to the study of structure, function, and differentiation, also provides important biologicals for the pharmaceutical industry, including viral vaccines, enzymes, hormones and antibodies. Microcarrier cell culture technology means that anchorage

7、 dependent animal cells are grown on the surface of small (approximately 150 micron diameter spheres which are maintained in stirred suspension cultures. This technology is replacing conventional monolayer cell culture methods as the extremely high surface area to volume ratio afforded by microcarri

8、ers is also used for: Routine cell culture New research applications High density culture Production culture volumes greater than 1000 litresSome of the advantages of the microcarrier system are: Efficient monitoring and culture control Reduced costs and contaminationApplicationsCytodex enables all

9、anchorage dependent animal cells to grow in suspension cultures, and is used in both batch and perfusion culture systems, as well as to increase the surface area of traditional monolayer cultures. In stirred suspension cultures, cells grow in a homogeneous environment where the culture parameters ar

10、e easily monitored and controlled. Cultures may be sampled, at will, to examine cell morphology and to determine cell viability. Microcarrier techniques are therefore the natural choice where cells are used for the production of biologicals. At the research level, applications with Cytodex include:

11、Studies of cell structure, function and differentiation Enzyme free subcultivation Implantation studies Harvesting of mitotic cells Light and electron microscopy Transportation and storageIndustrial scale applications utilizing Cytodex include the production of cells, cell products and viral vaccine

12、s (see Industrial applications, Page 11.34Cell attachmentIn order to culture anchorage dependent cells in vitro it is essential to know how the cells interactwith their environment. The cell membrane is surrounded by a "coat" called the glycocalyx(Figure 1. The glycocalyx consist of glycol

13、ipids, glycoproteins and transmembrane proteoglycans.A large number of these molecules contain sugars with a negative charge. So the net charge on theglycocalyx is negative. That means that if a culture surface is positively charged there will be anelectrostatic attraction between the cell and the c

14、ulture surface. It has been shown that the attachmentto gelatin microcarriers compared to charged carriers, is slower because the surface charge isnot optimal (1.Fig. 2. The subunit structure of a cell-surfacefibronectin receptor. By binding to fibronectinoutside the cell and to the cytoskeleton (vi

15、a theattachment protein talin inside the cell, the proteinserves as a transmembrane linker. The fibronectinreceptor belongs to a large superfamily of homologousmatrix receptors called integrins , most of whichrecognize RGD sequences in the extracellularproteins they bind.© Garland Publishing In

16、c.Fig. 1. Diagram of the cell coat (glycocalyx whichis made up of the oligosaccharide side chains ofglycolipids and integral membrane glycoproteins,and polysaccharide chains on integralproteoglycans. In addition, adsorbed glycoproteinsand absorbed proteoglycans (not shown contributeto the glycocalyx

17、 in some cells.© Garland Publishing Inc. Among the glycoproteins in the membrane, there isa family of heterodimeric glycoprotein receptorscalled integrins (Figure 2. These integrins help bindcells to mainly RDG sequences in extracellularmatrix (ECM proteins. The integrin receptorsrequire divale

18、nt ions such as Ca2+, Mg2+, Mn2+ forbinding to their ligands (2. This is the reason whyEDTA and Ca2+, Mg2+ free buffers are used whenharvesting cells in culture. The internal part of theintegrin receptor, interacts with the cytoskeleton,which makes it possible for the cell to spread outafter attachm

19、ent. The membrane integralproteoglycan molecules also bind to extracellularmatrix components. (Recently it has been shownthat membrane bound proteoglycans bind togrowth factors and thereby modulate their activity.Syndecan, a heparan sulfate proteoglycan binds tobasic FGF and Decorin and Betaglycan b

20、inds TGF-b (3. There are also unrelated transmembraneglycoproteins that bind to collagen.The ECM consist mainly of fibrous proteins embedded in a polysaccharide gel, which are secreted locally. The main ECM components are collagen, elastin, fibronectin, laminin, and the glycosaminoglycans. Glycosami

21、noglycans consists of long unbranched polysaccharide chains composed of repeating disaccharide units. A large number of the sugars are sulfated or contain carboxyl groups. This gives the GAG's high negative charges. The fact that the cells in vivo, are surrounded by hydrated sugar gels and fibro

22、us proteins is interesting when choosing materials for microcarriers. The fact that cells also interact with collagen have been utilized when designing Cytodex 3.The attachment process in vitro is summarized in Figure 3. Fig. 3. Simplified outline of steps involved in adhesion of animal cells to cul

23、ture surfaces. The whole involves divalent cations and glycoproteins (mainly fibronectin (Fn and vitronectin adsorbed to the culture surface. Under culture conditions the attachment glycoprotein originates from the serum supplement in the medium. MHS is synthesized by the cells. With anchorage-depen

24、dent cells proliferation occurs only after the spreading step. MHS multivalent heparin sulphate.5Requirements for an optimum microcarrier Properties and characteristicsTwo types of Cytodex are available to support the growth of all anchorage dependent animal cells for a multitude of applications, an

25、d the microcarriers are designed to meet the special requirements for this technology. The optimized bead size and density support maximum cell growth rate and cell yield. A biologically inert matrix provides a stable - but non ridgid - substrate for stirred cell67Size is based on diameter at 50% of

26、 the volume of a sample ofmicrocarriers (d50, or the range between the diameter at 5% and 95%of the volume of a sample of microcarriers (d5-95. Thus size iscalculated from cumulative volume distributions.Fig. 4. Scanning electron micrograph of BS-C1 cellsgrown on Cytodex 3. The cells were treated wi

27、thtrypsin for 3 minutes prior to preparation for SEM.cultures (80% of the bead volume is liquid i.e. the beads are small gel particles, from which cellsare easily harvested. Cytodex is transparent, allowing microscopic examination of attached cells.The physical characteristics of Cytodex are shown i

28、n Table 1.Table 1. Physical characteristics.Cytodex 1, formed by substituting a cross-linked dextran matrix with positively charged DEAE*(N,N-dimethylaminoethyl groups distributed throughout the matrix.Cytodex 3, formed by chemically coupling a thin layer of denatured collagen (Mr 60.000200.000, pig

29、 skin type 1 to the cross linked dextran matrix, is the microcarrier of choice for cells known tobe difficult to culture in vitro and particularly for cells with an epithelial-like morphology. Thiscollagen surface layer is susceptible to digestion by a variety of proteolytic enzymes and providesuniq

30、ue opportunities for harvesting cells from the microcarriers while maintaining maximum cellviability and membrane integrity. These issues may be critical in obtaining successful serialsubcultivation protocols required for scaling up culture volumes.Figure 5 is a schematic representation of the two t

31、ypes of Cytodex. Amersham Biosciences produces Cytodex inaccordance with current Good Manufacturing Practice (cGMP and every batch must conform tostringent specifications. Quality control tests are performed to satisfy both physiochemical andfunctional (i.e. cell growth properties (Figure 6. A certi

32、ficate of Analysis is available on request. + +Cytodex 1charges throughout matrixcollagen layercoupled tosurfaceCytodex 3cross-linked dextranCH CH23CH CH23.HC L 22CH CHN cross-linked dextranOHCH CH 2CH 2(-e LYS.COLLAGEN O NH Fig. 5. Schematic representation of the two types of Cytodex.Fig. 6. Each b

33、atch of Cytodex is fullyquality controlled, includingmonitoring of cell growth.Size* d (µm190175d 595 (µm147248141211Approx. area* (cm2/g dry weight4 4002 700Approx. no micro-carriers/gdry weight4.3 x 1063.0 x 106Swelling factor* (ml/g dry weight2015*in 0.9% NaCl.Instructions for usePrepar

34、ationCytodex, supplied as a dry powder, must be hydrated and sterilized before use. Autoclaving is the simplest method for sterilization; Cytodex is extremely stable, and if necessary can be autoclaved repeatedly (at least 5 cycles of 15 min, 115 °C, 15 psi or extensively (12 h, 130 °C, 27

35、 psi without affecting its performance. Prior to initiating a microcarrier culture, the pH and temperature of the culture medium containing the prepared microcarriers should be adjusted for optimal initial attachment of the cells to Cytodex. In order to achieve a good attachment the culture surface

36、has to be coated with fibronectin (15 ng/cm2 for BHK, 100 ng/cm2 for CHO or vitronectin, which are present in serum containing medium (10% v/v foetal calf serum contains 23 µg fibronectin/ml. For some epithelial, endothelial cells it might be necessary to coat the culture surface with laminin.

37、If serum free medium is used these proteins must be added, unless the cell line secretes them itself. In addition, the gas mixture in the culture vessel headspace should be equilibrated with the culture medium.InoculumThe state of the inoculum is the most critical factor affecting microcarrier cultu

38、res. The cells should be in logarithmic growth phase and in a good nutritional state, upon harvesting, and definitely in the resting G0 stage. This ascertains a short and efficient growth phase in the cultures. It is therefore essential to optimize the harvesting procedure, and the timing for harves

39、ting the inoculum culture, to maximize viability and yield.The cell inoculum should be introduced to the culture vessel only after equilibrium has been established. Good initial cell attachment is essential in order to obtain maximum final cell yields from microcarrier cultures. It is therefore of i

40、mportance to experiment with the initial conditions. Culture ProcedureThe exact culture requirements i.e., medium, serum and supplements depend on the cell type. The size of the final culture volume and the design of the culture vessel determine the optimal culture procedure. Batch type microcarrier

41、 cultures usually contain up to 5 grams of Cytodex per litre of culture volume. Fed batch and perfusion cultures can sustain higher cell densities (4 and Cytodex concentrations may be greater than 20 grams per litre.Monitoring cell growthRepresentative samples of the microcarrier culture may be with

42、drawn at any time during the culture period. The cells on the microcarriers can then be visualized directly by phase contrast microscopy or by light microscopy after the cells have been stained with, for example haematoxylin. The simplest and most accurate method for determining cell number is to us

43、e nucleus extrusion method where cells on microcarriers are lysed in 0.1 M citric acid. The released nuclei are stained in crystal violet and then counted in a haemacytometer (5. Another alternative is to measure intracellular metabolites released upon lysis (enzymes, ATP, for example.8Harvesting ce

44、llsThe most common methods for harvesting cells from Cytodex involve standard procedures employing proteolytic enzymes such as trypsin and collagenase. Pre-washing the confluent microcarriers with a solution of EDTA-PBS* (Ethylenediamine tetraacetic acid-phosphate buffered saline, prior to trypsiniz

45、ation, may improve the harvested yield of strongly adherent cells, by binding divalent ions and thereby destabilizing the binding strength between integrins and ECM proteins (see above. Especially when serum containing medium is used, it is necessary to efficiently wash away the protease inhibitors

46、which are present in the serum. Other-wise the enzyme activity goes down drastically!Special attention should be paid to optimizing and standardizing the harvesting procedure with respect to both the activity of and the time which the cells are exposed to, the enzyme solution (6, 7, 8. Other procedu

47、res include the use of chelating agents, exposure to hypotonic conditions, cold conditions, sonication or alteration of the surface tension of the culture medium (Figure 7.Scaling up by serial subcultivationSerial subcultivation of cells on microcarriers is the most cost-effective means for establis

48、hing animal cell culture production facilities, achieved using a series of culture vessels of increasing volume and capacity. The cells cultured in one vessel are subsequently used to inoculate the following larger vessel. This approach to scale up eliminates large numbers of culture vessels, such a

49、s roller bottles or multi-trays, otherwise required to obtain sufficient cells for inoculating the larger production scale fermenters. The protocol for serial subcultivation of cells from one culture of Cytodex to the next, should be firmly established at the small scale prior to scaling up to produ

50、ction culture volumes. Some cell types are capable of undergoing bead-to-bead migration with Cytodex, i.e. subcultivation can be performed without a harvesting step that employs proteolytic enzymes. In this case, scale up may be achieved simply by increasing the culture volume and adding more Cytode

51、x. Fig. 7. The modified Vibromixer vessel used by van Wezel et al., for concentrating, washing and trypsinizing the cells, separating the cells from the used microcarriers.Fig. 8. Europes latest facilities for the production of polio virus vaccine using Cytodex microcarriers. Courtesy of Rijksinstit

52、uut voor Volksgezondheid en Milieuhygiëne (RIVM, The Netherlands.9Suggestions to increase or facilitate the migration includes the stirring speed, which can be increased (cells detach during cell division and re attatches to fresh beads, the medium composition can be manipulated, increase phosphate and decrease Ca2+, Mg2+, Mn2+ concentrations, less f