1.4.4 Studies on cell function,metabolism and differentiation Micr h nient culture urfaces in r any cell biology sitios in a h neous cultur e system vides ities for studie of coll f LeLopRatoitieic make it easter to manipulate and observe ells Whe d with traditional r whic vide for t for y densities and when microcarriers are packed together a culture can be achieved. atible with cell function and differentiation in vitro and a wide variety of different studies have been re orted.The choice of the most suitable microca rier is described in section 2.5 and for most studies with differentiating systems Cytodex 3 is the microcarrier of choice(table 5).Several examples serve to illustrate the use of microcarriers in cell biology studies. Pawlowski et al(61)used Cytodex to study the differentiation of chick embryo skeletal muscle cells.Normal myogenesis occured on the microcarriers which were also used for mic roscopy studies (plate 1).After 4 days of culture 62%of the microcarriers had myotubes with extensive myofibril formation(61). With an even more sensitive cell system Moser and Stoffels(62)studied the differentiation of newborn rat heart muscle cells.The microcarrier method provided homogeneous and easily manipulated cultures.The heart cells spread and proliferated on the microcarriers and expressed pacemaker membrane properties. Between 20-30%of confluent monolayers on the microcarriers exhibited spontaneous beating activity(62). The release of insulin from foetal rat pancreas islet cells growing on Cytodex has been studied by Bone et al(63.64).These studies demonstrated that Cytodex is suitable for maintaining highly specialized endocrine cells in culture.The Dissociation Fig.14.The s ure of prim ier (3-5 days) 心 er sup on system Monitor hormone secretion 22
22 1.4.4 Studies on cell function, metabolism and differentiation Microcarriers can be used as convenient culture surfaces in many cell biology studies. The ability to culture cells at high densities in a homogeneous culture system provides unique opportunities for studies of cell function, metabolism and differentiation. In addition microcarriers make it easier to manipulate and observe the cells. When compared with traditional monolayer techniques which only provide for two-dimensional cultures, the microcarrier system allows for very high culture densities and when confluent microcarriers are packed together, a three-dimensional culture can be achieved. Cytodex microcarriers are compatible with cell function and differentiation in vitro and a wide variety of different studies have been reported. The choice of the most suitable microcarrier is described in section 2.5 and for most studies with differentiating systems Cytodex 3 is the microcarrier of choice (table 5). Several examples serve to illustrate the use of microcarriers in cell biology studies. Pawlowski et al (61) used Cytodex to study the differentiation of chick embryo skeletal muscle cells. Normal myogenesis occured on the microcarriers which were also used for microscopy studies (plate 1). After 4 days of culture 62% of the microcarriers had myotubes with extensive myofibril formation (61). With an even more sensitive cell system Moser and Stoffels (62) studied the differentiation of newborn rat heart muscle cells. The microcarrier method provided homogeneous and easily manipulated cultures. The heart cells spread and proliferated on the microcarriers and expressed pacemaker membrane properties. Between 20-30% of confluent monolayers on the microcarriers exhibited spontaneous beating activity (62). The release of insulin from foetal rat pancreas islet cells growing on Cytodex has been studied by Bone et al (63,64). These studies demonstrated that Cytodex is suitable for maintaining highly specialized endocrine cells in culture. The Dissociation Micro carrier culture (3-5 days) 95% of cells attach Hypothalamic releasing factors Chromatographic column superperfusion system Monitor hormone secretion Fig. 14. The superperfusion culture of primary rat pituitary cells growing on Cydodex 1 microcarriers. The scheme is based on studies by Smith and Vale (65, 66)
ed t for affs and h.M.A.and of the authors and publisher) microcarriers provided a method for the uniform suspension culture of functioning pancreas cells and allowed for easy manipulation of the cells(64).The pancreas cells sustained synthesis and release of insulin during a 7 day growth period on the microcarriers (plate 3)and the release could be modulated by glucose and stimulated with theophylline(64). Microcarriers have been used in novel culture systems to study the function of differentiated cells.Smith and Vale(65.66)have developed a superperfusion column technique for the study of rat anterior pituitary cells and the modulation of pituitary secretions by gonadotrophins and cocarcinogens(fig.14).The system provided responsive and well-defined high density cultures which maintained the ability to secrete hormones fore long periods of time.The dissociated pituitary cells boypomcor (epmeth c attached and the culture system could be used to study transient phenomena and desensitization(65). A variety of other differentiated cells have been studied using Cytodex microcarriers. Ryan et a (67)developed a microcarrier culture system for studying the role of bovine pulmonary endothelial ce s and C.Busch (pers.comm. 187)has used Cytodex 3 in studies of endothelial cells from brain capillary and pulmonary artery (plate 2).Porcine thyroid cells cultured on Cytodex ex ibited an epithelial morphology and were capable of releasing thyroglobulin (68). 23
23 microcarriers provided a method for the uniform suspension culture of functioning pancreas cells and allowed for easy manipulation of the cells (64). The pancreas cells sustained synthesis and release of insulin during a 7 day growth period on the microcarriers (plate 3) and the release could be modulated by glucose and stimulated with theophylline (64). Microcarriers have been used in novel culture systems to study the function of differentiated cells. Smith and Vale (65,66) have developed a superperfusion column technique for the study of rat anterior pituitary cells and the modulation of pituitary secretions by gonadotrophins and cocarcinogens (fig. 14). The system provided responsive and well-defined high density cultures which maintained the ability to secrete hormones fore long periods of time. The dissociated pituitary cells attached to the Cytodex microcarriers (fig. 15) and remained responsive to hypothalamic releasing factors (65,66, fig. 16). Approximately 95% of the cells attached and the culture system could be used to study transient phenomena and desensitization (65). A variety of other differentiated cells have been studied using Cytodex microcarriers. Ryan et al (67) developed a microcarrier culture system for studying the role of bovine pulmonary endothelial cells and C. Busch (pers. comm., 187) has used Cytodex 3 in studies of endothelial cells from brain capillary and pulmonary artery (plate 2). Porcine thyroid cells cultured on Cytodex exhibited an epithelial morphology and were capable of releasing thyroglobulin (68). Fig. 15. Primary rat interior pituitary cells attached to Cydodex 1 microcarriers and incubated for 5 days after dissociation. (Smith, M.A. and Vale, W.W. Endocrinol. 107 (1980) 1425, by kind permission of the authors and publisher.)
40 0 200 100 03691215182124 0 3691215182124 Hours Fig 16.Res ed in th ig.14 and 15 min pulses of 30 nM GnRH every 2 h.Flow rate was0.2 mmin and fractions were Vale.W.Wnd().bykind pro th (Sr and pu Vosbeck and Roth(69)used microcarrier culture to study the effects of different treatments on intercellular adhesion.Confluent monolayers of cells were cultured on microcarriers and intercellular adhesion was examined by studying the binding of RP-labelled cells to the monolavers (69). Lymphocytes have been grown on microcarriers for studies of stimulation(43). Cytodex 1 alone was not mitogenic for lymphocytes but potentiated stimulation by Con A(fig.17).The microcarriers have been used to study the relationship between anchorage,cell density and stimulation of lymphocytes (43). Microcarriers are also used in studies of animal cell plas membranes.Lai et al (70)used Cytodex to study the influence of adhesion on the fluidity on Chinese prreeo clre amdhe ance technique it microcarriers in suspension culture.Cytodex microcarriers are compatible with spin- labelling and provided a technique whereby cells cou Id be easily transferred and assayed without removal from the culture surface (70) Microcarrier culture can als be used for don fr 7.7 anes with procedure is suitable for cells apable of atta s are followed bybrief d to attach and spr on tn carri to disrupt the cells The cell deb s is then removed and memb microcarriers can be use enzymes(71) 24
24 Vosbeck and Roth (69) used microcarrier culture to study the effects of different treatments on intercellular adhesion. Confluent monolayers of cells were cultured on microcarriers and intercellular adhesion was examined by studying the binding of 32P-labelled cells to the monolayers (69). Lymphocytes have been grown on microcarriers for studies of stimulation (43). Cytodex 1 alone was not mitogenic for lymphocytes but potentiated stimulation by Con A (fig. 17). The microcarriers have been used to study the relationship between anchorage, cell density and stimulation of lymphocytes (43). Microcarriers are also used in studies of animal cell plasma membranes. Lai et al (70) used Cytodex to study the influence of adhesion on the fluidity on Chinese hamster ovary cell plasma membranes. By using electron spin resonance technique it was possible to compare cells growing in free suspension culture and attached to microcarriers in suspension culture. Cytodex microcarriers are compatible with spinlabelling and provided a technique whereby cells could be easily transferred and assayed without removal from the culture surface (70). Microcarrier culture can also be used for the isolation of plasma membranes with less than 1% contamination from internal membrane markers (71). The procedure is suitable for cells capable of attachment. Cells are first allowed to attach and spread on the microcarriers. Hypotonic lysis is followed by brief sonication to disrupt the cells. The cell debris is then removed and membranes attached to the microcarriers can be used directly for assays of membrane-associated enzymes (71). a b LH secretion (ng/min) Hours FSH secretion (ng/min) 100 200 300 400 03 69 12 15 18 21 24 03 69 12 15 18 21 24 25 50 75 100 Fig. 16. Response of rat anterior pituitary cells growing on Cydodex 1 microcarriers to pulses of gonadotropin-releasing hormone (GnRH). The cells were cultured in the system illustrated in fig. 14 and were exposed to 15 min pulses of 30 nM GnRH every 2 h. Flow rate was 0.2 ml/min and fractions were collected every 20 min. a. Secretion of lutenizing hormone. b. Secretion of follicle stimulating hormone. (Smith, M.A. and Vale, W.W., Endocrinol. 108 (1981) 752, by kind permission of the authors and publisher.)
加An arriers.Th (Sundqvis ek.and 573.by kin uth the publisher 05101520253050 g Con A/m 1.4.5 Proteolytic enzyme-free subcultivation and cell transfer In many studies it is important to be able to harvest or transfer cultured cells out using proteollytic enzyme or chelati ng agents er cell viability and the integrity of the plasma membranes.By using microcarriers it is e to sut re cel c enzymes or also provide conver aces for cells can E from c ssel to culture ves I or used directly for experiments with having to be removed from the microcarriers microcar flask microcarrie th we hi onto yers of (72).T me thod of ansfo proteolytic are norm nally sensitive to treatment s(67 A variant of this technique is to allow cells to arri si andt Thilly 6) tial F CHO ells by dilu nfluent mi riers The tra sfer of cells betw anced ith low Cazt ion (6)Ne lso be added to confluent microc eriods of virus Manou t al (55)used this tech t ring pe carrier cultures rpblasts by allowing conet micrcarriers totwith ne microcarriers and after ation of cells results in inoculation of the new microcarriers (P.Talbot. oers.com nm.188) 25
25 1.4.5 Proteolytic enzyme-free subcultivation and cell transfer In many studies it is important to be able to harvest or transfer cultured cells without using proteollytic enzymes or chelating agents. Such agents often alter cell viability and the integrity of the plasma membranes. By using microcarriers it is possible to subculture cells or scale-up cultures without using proteolytic enzymes or chelating agents. Microcarriers also provide convenient surfaces for cell growth and cells can be transferred from culture vessel to culture vessel or used directly for experiments without having to be removed from the microcarriers. Horst et al (72) and Ryan et al (67) observed that cells can migrate between microcarriers and the surfaces of cell culture flasks or Petri dishes. Cytodex microcarriers were allowed to settle onto monolayers of mouse fibroblasts and cells migrated onto the microcarriers which could then be transformed to another culture vessel (72). This method of transfering cells has also been used for bovine pulmonary artery endothelial cells; such cells are normally sensitive to treatment with proteolytic enzymes (67). A variant of this technique is to allow cells to migrate from confluent microcarriers onto new microcarriers. Crepsi andt Thilly (6) could maintain prolonged periods of exponential growth of CHO cells by diluting confluent microcarrier cultures and adding new microcarriers. The transfer of cells between microcarriers was enhanced by using a medium with low Ca2+ concentration (6). New microcarriers can also be added to confluent microcarrier cultures during periods of virus production and Manousos et al (55) used this technique to cause a new wave of cell proliferation and production of oncornavirus. It is also possible to scale up microcarrier cultures of human fibroblasts by allowing confluent microcarriers to settle with new microcarriers and after a few hours, migration of cells results in inoculation of the new microcarriers (P. Talbot, pers. comm., 188) cpm x 10-3 µg Con A/ml 0 5 10 15 20 25 30 50 10 20 30 40 50 60 70 80 Fig. 17. The stimulation of human lymphocytes by Con A in the presence (❍) or absence (●) of Cydodex microcarriers. The microcarriers alone were not mitogenic. (Sundqvist, K. and Wagner, L., Immunology 43 (1981) 573, by kind permission of the authors and the publisher.)
The suitability of this technique is limited by the mobility of cells.Some types of cells,e.g.hepatocytes,exhibit only limited mobility and do not migrate between microcarriers and other culture surfaces.The chance of cell transfer between microcarriers is increased by allowing the culture to remain static for several hours. Occasional stirring should eliminate any tendency for the microcarriers to aggregate. Microcarriers also have other applications in the transfer of cells.For example ete mre the he p b simple differential sedimentation (H.Slater,pers.comm.,189).The macrophages attached to the microcarriers can then be transfered to other culture vessels for study. Microcarriers can also be used for cloning cells.Cultures can be inoculated with approximately one cell/microcarrier and after allowing time for cell attachment ogaeaaeO8oesped品rayege ed culture surface.Similarly confluent microcarriers can be embedded in semi-solid medium to form feeder layers. 1.4.6 Microscopy Cytodex microcarriers can be used as cell culture substrates for a variety of microscopy studies using standard techniques such as scanning electron microscopy (f1.plates 1.).transmission electron microscopy ()and different typesof light microscopy illumination and cytochemistry (plates 2.3.5-7.9).The advantages of using microcarriers for microscope is that such culture substrates are easy to manipulate and cells do not need to be harvested when embedding techniques are used.The dextran-based matrix of Cytodex microcarriers(section 2.1)can be penetrated by the usual embedding media before sectioning.By using confluent microcarriers transverse sections through cells adhering to the culture substrate can be readily obtained (fig.3) Routine samples from microcarrier cultures can be processed for detailed microscopical examination and cultures containing many coverslips can be avoided. Similarly samples of experimental cultures can be processed for microscopy without requiring large numbers of cells. Details of microscopy with Cytodex microcarriers can be found in section 3.6. 1.4.7 Harvesting mitotic cells e tm een mue ached only weakly to cell culture surfaces and can be detached by shaking (75).The use of monolayer culture vessels for this technique is limited by the small surface area for cell growth and microcarriers provide the large surface area necessary for recovering high yields of mitotic cells. 26
26 The suitability of this technique is limited by the mobility of cells. Some types of cells, e.g. hepatocytes, exhibit only limited mobility and do not migrate between microcarriers and other culture surfaces. The chance of cell transfer between microcarriers is increased by allowing the culture to remain static for several hours. Occasional stirring should eliminate any tendency for the microcarriers to aggregate. Microcarriers also have other applications in the transfer of cells. For example, Cytodex can be incubated with peritoneal fluid and after 10 min macrophages adhere to the microcarriers and can be separated from the other peritoneal cells by simple differential sedimentation (H. Slater, pers. comm., 189). The macrophages attached to the microcarriers can then be transfered to other culture vessels for study. Microcarriers can also be used for cloning cells. Cultures can be inoculated with approximately one cell/microcarrier and after allowing time for cell attachment those microcarriers bearing only one cell can be transfered by Pasteur pipette into cloning wells. In this way the microcarriers provide an easily manipulated culture surface. Similarly confluent microcarriers can be embedded in semi-solid medium to form feeder layers. 1.4.6 Microscopy Cytodex microcarriers can be used as cell culture substrates for a variety of microscopy studies using standard techniques such as scanning electron microscopy (fig. 1, plates 1,4,8), transmission electron microscopy (fig. 3) and different types of light microscopy illumination and cytochemistry (plates 2,3,5-7,9). The advantages of using microcarriers for microscope is that such culture substrates are easy to manipulate and cells do not need to be harvested when embedding techniques are used. The dextran-based matrix of Cytodex microcarriers (section 2.1) can be penetrated by the usual embedding media before sectioning. By using confluent microcarriers transverse sections through cells adhering to the culture substrate can be readily obtained (fig. 3) Routine samples from microcarrier cultures can be processed for detailed microscopical examination and cultures containing many coverslips can be avoided. Similarly samples of experimental cultures can be processed for microscopy without requiring large numbers of cells . Details of microscopy with Cytodex microcarriers can be found in section 3.6. 1.4.7 Harvesting mitotic cells Microcarrier culture provides an efficient method for harvesting mitotic cells (73,74). The technique is based on the observation that mitotic cells are attached only weakly to cell culture surfaces and can be detached by shaking (75). The use of monolayer culture vessels for this technique is limited by the small surface area for cell growth and microcarriers provide the large surface area necessary for recovering high yields of mitotic cells