Microcarrier culture of vascular endothelial cells on solid plastic beads

Copyright @ 1981 by Academic Press. Inc. All rights of reproduction in any form reserved 0014.4827181/080367-10$02.00/O

Experimental Cell Research 134 (1981) 367-376

MICROCARRIER

CULTURE

CELLS

OF VASCULAR

ON SOLID

PLASTIC

ENDOTHELIAL

BEADS

PETER F. DAVIES Vuscular Pathophysiology

Laboratory. Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 021 IS, USA

SUMMARY The culture of vascular endothelial cells on solid plastic beads is described. A greater than 30-fold increase in cell numbers was achieved in stationary culture medium. The inclusion of fibroblast growth factor slightly improved the rate of growth from low densities. Addition of fresh beads to colonized beads resulted in colonization of the newly introduced microcarrier. In common with the behaviour of endothelium in conventional culture, the cells cultured on beads changed from a fusiform to a polygonal shape after reaching confluence. Cell proliferation was also observed by [3H]thymidine autoradiography of DNA. The fraction of radiolabelled nuclei declined at confluence on each bead, indicating density-inhibition of growth. By electron microscopy, the cells displayed the typical ultrastructural appearance of endothelium. Following transfer of colonized beads.to a chromatography column with slow perfusion of the bead bed, cell viability was maintained over a 24 h period and proportional synthesis of prostaglandin I2 upon stimulation by ionophore A23187 was demonstrated. This simple microcarrier technique allows the generation of large numbers of vascular endothelial cells for subcellular fractionation with economical use of space and medium. When set up as a perfused bead bed, it offers possibilities for the short-term collection of concentrated endothelial metabolites.

Vascular endothelial cell culture is now firmly established as a valuable research approach to the pathophysiology of blood vessels [I, 21. Endothelial cells of arterial and venous origin and derived from a variety of species [l-5] form strictly contactinhibited monolayers which are unresponsive to the effects of known growth factors [6-81. The substratum routinely employed for endothelial tissue culture is the flat plastic surface of Petri dishes and flasks in which a confluent saturation density of -1.5~105 cells/cm2 is achieved [l, 2, 91, a density which corresponds closely to that observed in arterial tissue in vivo. For studies involving subcellular fractionation of endothelial cells, the generation of large

quantities of cells is limited by the surface area available in plastic flasks. A large increase in surface area for cell growth with economical utilization of space and materials can be accomplished by culture of the cells upon spherical beads. The so-called microcarrier system devised by van Wezel [lo] using modified DEAESephadex has been effectively utilized in the production of a number of viral vaccines [ 11, 121 and in the production of interferon by diploid fibroblasts [ 131. Recently a solid plastic bead with properties similar to tissue culture plastic ware has become commercially available (Biosilon, A/S Nunc) [14] and appears particularly suited to the growth of normal diploid [15]

368

P. F. Davies posure to crude collagenase (CI. histolyticum, Type I, 1 mg/ml; Worthington Biochemicals, Freehold, NJ) at 37°C. The swab, with attached cells, was rotated in a 25 cm* Falcon flask containing tissue culture medium (TCM) to release the sheets of endothelial cells. The detached cells became adherent to the flask’s surface as sheets and islands of endothelium. A confluent monolayer was rapidlv established in TCM containing 5% bovine plasma-derived serum (PDS), prepared as described by Vogel et al. [17]. The PDS inhibited the growth of occasional contaminating smooth muscle cells without affecting the proliferation of endothelial cells. Serial passage of endothelial cells (split ratio 1 : 3) eliminated, bv dilution. any contaminating smooth muscle cells. Beyond the 3rd passage, cells were fed every other day with TCM containing 10% calf serum (M.A. Bioproducts, Bethesda, Md). The basal medium was Dulbecco’s modified Eagle medium supplemented with 2 pmol/ ml glutamine and 100 units each of penicillin and streptomycin per ml.

Cells plated on beads Fig. 1. (a) Colonization of plastic beads by arterial endothelium; (b) dissociation of cells from the bead surface by trypsin. Phase contrast. Focal plane at bead edge. X 100.

and transformed [16] cells. In this communication, the culture of bovine arterial endothelial cells on solid plastic beads using simple apparatus is described. The technique appears to be particularly suited to the production of large, homogeneous cultures of endothelium which retain the morphological and growth characteristics of their conventionally-cultured counterparts. Furthermore, the non-porous nature of the beads eliminates adsorptive surface area within the bead, a property which may be advantageous for the collection in the culture medium of specific endothelial metabolites. MATERIALS Isolation

AND METHODS

of endothelial

cells

Bovine arterial endothelium was obtained by a modification of the method described by Gimbrone et al. [I]. A sterile cotton swab was gently applied to the luminal surface of calf aorta following a IO min exExp Cell Res 134 (1981)

Experiments were conducted using a microcarrier support system of negatively-charged spherical plastic beads (Biosilon) manufactured by A/S Nunc, Denmark (Vanguard International, Neptune, NJ). The published diameter range is 160-300 irn [14] although the distribution of beads used in the studies described here tended towards the smaller diameter (measured mean diameter 205 pm). Cells were allowed to attach to the beads in the absence of flow after incubating the beads with medium containing calf serum. This probably allowed the bead surface to be coated with plasma cold-insoluble globulin (CIg) and appeared to be a crucial step allowing attachment. In contrast, cells in serum-free medium attached poorly and failed to flatten. For plating, beads were incubated with TCM for 30 min at 37°C in a plastic Petri dish, the surface of which had been treated with the inert polymer, hydroxyethyl methacrylate (Hydron Laboratories, New Brunswick, NJ: 1.2% solution in ethanol), or siliconized water repellent (2% solution of dimethyldichlorosilane in carbon tetrachloride; Fisher Chemical Co., Fair Lawn, NJ). Failure to treat the dish surface greatly reduced the attachment of cells to the beads because of competition for attachment to the dish surface. For the growth studies, 24 mg beads were added to 16 mm-diameter tissue culture wells (Costar, Cambridge, Mass.). Cell inocula of various sizes (3-40X 10“ cells) in 1 ml of TCM were introduced and cell counts were determined periodically thereafter. In a scaled up version for the production of about lOa cells, a 10 cm Petri dish and 2 g beads ( -3.7~ 105beads/s) corresoondina to a growth area of 970 cm2 with a cell inoculum >f 3x-106 in 10 ml TCM was satisfactory. The unattached cells and beads were then gently mixed with a siliconized glass rod and left undisturbed. The cells attached to the beads and flattened within 2-3 h. The medium was changed every 2 days. All cultures were incubated at 37°C in a humidified 5 % Con-air atmosphere. To determine cell and bead numbers and cell: bead ratios, either small aliquots of beads were sam-

Microcarrier

101

0

369

culture of endothelium

IIIIII

2

II

4

6

8

I

IO

days In culture Fig. 2. Growth curves of microcarrier-cultured endothelium in absence of flow and in (a) the absence; or (6) presence of fibroblast growth factor (FGF; 100 nglml). To 24 mg beads were added a range of

cell inoculum densities (cell : bead ratios l-25) in 10% calf serum. At various intervals, the cell : bead ratio was measured as described in the text.

pled from a large culture and transferred to a siliconised test tube, or entire wells were sacrificed. In both cases the medium was removed, the microcarrier system rinsed twice with phosphate-buffered saline (PBS) deficient in Ca2+ and Mg*+ to remove serum and unattached cells, then incubated briefly with a solution of EDTA : trypsin (0.2 % : 0.5 %). The detached cells and denuded beads were suspended by vigorous pipetting and aliquots were removed for cell counting by hemocytometry. The number of beads

was determined by transfer of an aliquot to a clean plastic dish where the total number was determined with the assistance of an ocular scale.

Cytology and autoradiography Cells bound to the beads were fixed with ethanol: glacial acetic acid (3 : 1) for 5 min at room temperature, rinsed with PBS, transferred to a glass slide and stained with hematoxylin. For autoradiograuhv, the microcarrier svstem was incubated with TCM containing 0.5 &i [methyVH]thvmidine oer ml (6.7 CilmM) for 24 h at 37°C. The cultures were rinsed with PBS and fixed with acetic acid : ethanol. The bead suspensions in water were then applied to a microporous filter (0.4 pm; Millipore Corp., Bedford, Mass.) held in a standard filter holder. The beads became embedded on the filter which served as a suitable support during subsequent manipulations. The filters were coated with a 50% solution (aq) of NTB2 emulsion (Eastman Kodak, Rochester, NY), exposed in the dark at 4°C for 10 days, then developed and fixed. The beads were photographed both before and after hematoxylin staining and after rendering the filter transparent with glycerol.

Ultrastructure Fig. 3. Appearance of microcarrier system at a cell :

bead ratio of 120. Hematoxylin stain. x 120.

Cells were prepared for transmission electron microscopy (TEM) in situ on the beads. The cultures Exp CeNRes 134 (1981)

370

P. F. Davies 160’

-

-0 : qooc = d

Ocells

per bead

Fig. 4. Distribution of cells within bead population with time in culture. Cells were plated at an average cell : bead ratio of 20. The same cultures were sampled and scored on days I, 4 and I I.

were rinsed twice with PBS and fixed for 1 h at 4°C in 3 % glutara)dehydeO. 1 M cacodylate buffer pH 7.4 containing 0.05 % CaCI,. After a 5 min wash with 0. I5 M cacodylate buffer, the cultures were incubated at 4°C for 1 h in 2% osmium tetroxide in 0.1 M cacodylate buffer then washed three times (5 min each) with buffer. The cultures were then incubated for 30 min with I % tannic acid in 0.5 M cacodylate buffer and for 5 min with 1% sodium sulphate in 0.05 M cacodylate buffer before dehydration through a graded series of ethanol. Some cultures were also stained with toluidine blue. The beads were floated on Epon in a BEEM capsule filled to a convex meniscus. The Epon was cured for 24 h, sectioned and stained with uranyl acetate and lead citrate then examined in a Philips EM 200 microscope. For scanning electron microscopy (SEM), cultures were rinsed with PBS, fixed with 3% glutaraldehyde in cacodylate buffer for 1 h at 4”C, rinsed repeatedly with deionised water, then either air-dried or dehydrated by a graded series of ethanol solutions. The results of either dehydration process were similar. The beads adhered to sticky copper tape applied to the surface of a standard SEM stub. The specimens were then coated with gold on a Hummer IV sputtercoater (Technics, Alexandra, Va) and viewed in an AMR 1000scanning electron microscope.

Stimulation of prostacyclin (PGIZ) synthesis 2.1 X 10’ cells maintained at 37°C on 0.6 g beads were transferred to a chromatography column (4.4 cm 0; Amicon, Lexington, Mass.) and arranged in a shallow bed (height ~0.1 cm) between two column adjusters. The bed was perfused with tissue culture medium at a rate of I ml/min (see fig. 11). The perfusion was Exp Cell RPS 134 (1981)

days

after

addition of

fresh

beads

Fig. 5. Colonization of beads freshly introduced into the established microcarrier system. On day 0, a quantity of beads equal to the established cultures was introduced thereby halving the cell : bead ratio. On days 1 and 8, the cell : bead ratio was determined.

stopped while I ml serum-free medium containing IO PM ionophore A23187 (Sigma, St Louis, MO.) was injected into the bead bed. After 15 min, the medium was drained from around the bead bed and collected for radioimmunoassay of 6-Keto-PG-Fla, the stable derivative of prostacyclin, according to the methods of Czervionke et al. [18]. Injection of serum-free medium alone acted as control.

RESULTS Cultured bovine aortic endothelial cells examined in the 6th-30th subculture readily adhered to the microcarrier system and flattened out within 2 h under static conditions. From studies of endothelial growth on plastic surfaces, a saturation density of -1 SX 105/cm2 has been repeatedly observed. The mean surface area of Biosilon plastic beads calculated from the measured diameter was 1.32~10~~ cm*. Thus the maximum theoretical cell : bead ratio in the microcarrier system is 198 cells/bead. In our experiments under static conditions, cells readily grew from a cell : bead ratio

Microcarrier

Fig. 6. (a) Fusiform shape of growing endothelium.

Mean cell : bead ratio 110. (b) Polygonal shape of confluent, growth-inhibited endothelium. Mean cell : bead ratio 175. Hematoxylin stain. (c) [3H]Thymidine DNA autoradiography of subconfluent endothelium. La-

as low as 3.5 (2.8~10~ cells/cm2 of growth surface) to close to saturation density (1.3~ loj cells/cm2; -180 cells/bead), an increase in cell numbers of almost 50-fold. The microcarrier system colonized by vascular endothelium is shown in fig. lo by phase contrast. Although cells could be observed under phase contrast only by focusing on the periphery of the beads, a reliable estimate of colonization was obtained because the orientation of the beads was random. This is clearly demonstrated in fig. 1b which shows the appearance of the beads following removal of endothelium by trypsin treatment. The influence of inoculation density upon growth in the microcarrier

culture of endothelium

371

belled nuclei stain black. (d) [3H]Thymidine autoradiography of confluent endothelium at saturation density. Few cells (3) are labelled (nrrm,s). (n. b) X250; (c) x 150; (d) x 120.

system under static conditions is shown in fig. 2~7. The inclusion of fibroblast growth factor (100 rig/ml, FGF; KOR Biochemicals, Cambridge, Mass.) resulted in a more rapid attainment of confluent densities from sparse inocula (fig. 2b). The inoculation of cells at cell: bead ratios below about 10 resulted in unequal distribution of cells throughout the bead population. This was directly observed even after extensive growth to a mean cell : bead ratio of 120 (fig. 3), where some beads carried a low cell population, whereas others carried confluent cultures. A semi-quantitative evaluation of the distribution of cells with time in culture is presented in fig. 4, and demonExp Cell Res 134 (1981)

372 P. F. Davies

Fig. 8. Transverse section of confluent endothelium. Light microscopy. Toluidine blue stain. x400. Fig. 7. SEM of confluent endothelium. Cells remained flat except for an occasional mitosis (arrow). X 185.

ing endothelium. Later, at confluence, the cells changed to a polygonal shape typical strates the existence of a few heavily colo- of density inhibition of growth observed on nized cells soon after plating. As the num- flat plastic surfaces. By C3H]thymidine autobers of cells/bead shifted to higher figures radiography, a proportion of cell nuclei by day 11, few beads remained unpopu- were labelled in the subconfluent cultures, lated. This raised the question as to wheth- in contrast to very low labelling several er naked beads could become colonized days after confluency (fig. 6c, d). As is from a confluent bead population. To test apparent from the SEM observations (fig. this hypothesis, microcarrier cultures of 7), endothelium forms a rather flat monoendothelium were divided into two groups. layer on the beads except where cells are One group was seeded with an equal in mitosis (arrow, fig. 7). In transverse amount of fresh beads and the cell : bead section, the close apposition of cells at conratio was measured daily thereafter. Fig. 5 fluence is apparent (fig. 8), and by TEM shows that 83 % of the original cell : bead (figs 9, lo), the appearance of the cells ratio was re-established 8 days after halv- was entirely consistent with endothelial ing it by addition of naked beads. The % morphology as observed on plastic Petri colonization never returned to lOO%, even dishes. The feasibility of setting up a bead bed after 2 weeks, a few beads always remainusing commonly available materials through ing uncolonized. The colonization of fresh beads was also confirmed by staining and which tissue culture medium could be perlight microscopy. Thus, continuous culture fused was investigated. Two gram of beads of endothelium may allow repeated passage colonized by just under lo8 cells (cell : bead ratio= 124) were carefully transferred to a of the cells to be avoided. siliconized glass column of internal diameThe morphological features of proliferating and confluent endothelium on micro- ter 1 cm (Amicon Corp., Lexington, Mass.). carriers are shown in fig. 6a, 6. The cells By lowering a column adjuster, the bead assumed the typical fusiform shape of grow- bed was enclosed between two supports, Exp Cd Res 134 (1981)

Microcarrier

Fig. 9. TEM of endothelial cell in confluent monolayer on plastic bead. The dark colour associated with the lower surface of the cell occurred with tannic acid staining. X 1500.

culture of endothelium

373

Fig. 10. TEM of similar cell as shown in fig. 9 to show a normal complement of intracellular organelles, attachment sites and plasmalemmal invaginations. x5 250.

30 ml TCM (fig. 11). In this system the extracellular space in the bead bed was measeach of which accommodated TCM entry ured as 0.7 ml. After 24 h, the beads were and exit tubing (1 mm internal diameter). washed, trypsinized and the cell : bead ratio TCM was perfused in a retrograde fashion and cell viability (dye exclusion) were dethrough the bead bed at a flow rate of 1 termined to be 110 and 93% respectively. ml/min and was recycled to a container of A 4.4 cm diameter glass column has also

Table 1. Stimulation” of prostaglandin I2 (PGZd synthesis in endothelial in monolayer and on solid plastic beads

cells cultured

PGI, detected as stable 6-keto-PG . Flor by radiommunoassay

A. Monolayer (plastic flask) (7.9X 106cells) B. Microcarrier (plastic beads) (2.1X 10’ cells)

nglculturell5 min

ng/106 cells/lS min

DMEM alone

DMEM+A23187

DMEM+ A23 187

1.63

48.6

6.1

3.40

104.2

5.2

a PGI, synthesis was induced by 10 WM A23187 dissolved in DMSO (final concentration 0.1%) in Dulbecco’s Modified Eagle medium (DMEM). Exp Cell Res 134 (1981)

374

P. F. Davies PGIz was collected in 1 ml of medium as compared with 3 ml of medium from the monolayer, thus effecting a 65fold concentration of PGI, (104 rig/ml from the beads and 16 nglml from the monolayer).

DISCUSSION Arterial endothelial cells were cultured on the surface of solid plastic beads under static or perfusion-flow conditions. The morphological characteristics and growth patterns were similar to those of endothelium cultured on a conventional flat plastic surface. The demonstration that naked 4 air-CO2 beads are readily colonized by cells derived per Lf . 37O from an existing microcarrier population Pump suggests that continuous culture of endoFig. II. Schematic diagram of bead bed perfusion thelium without exposure to trypsin is possystem for the short-term maintenance of cultured endothelium. Tissue culture medium (TCM) was sible. The generation of large quantities of drawn from a reservoir, A, at 10 ml/min via a per- cells is useful for studies involving cell fusion pump. 90% of the TCM was recycled directlv to the reservoir at the first flow valve, &, which was fractionation and in addition allows the easy adjusted to direct 1 ml/min through the bead bed, transfer of cells to other culture or assay C. TCM from the bead bed can be collected, D, or Furtherreturned to the reservoir. Local injection into the bead systems without trypsinization. bed of agents of interest can be accomplished via a more, maintenance of the microcarrierLuer-type valve adjacent to the bead bed. endothelial system in a bed perfused by tissue culture medium may be useful for the been used in this system in order to reduce harvest of endothelial-specific molecules the bed height and thus minimise concen- synthesized by, or modified within the tration gradient effects within the bed. Us- cells. In 1967, van Wezel demonstrated the ing such a bead bed, synthesis of prostacyclin (PGI,) was stimulated 30-fold by 10 feasibility of culturing various cell types on PM A23187 [ 19, 201, (table 1). Injection of DEAE-Sephadex spheres [lo]. A major serum-free medium into the bead bed for 15 technical improvement was the titration of min at 37°C resulted in a basal PGI, syn- the Sephadex beads to low anion exchange thetic rate of 3.4 ng by 2~10~ cells. This capacity [21, 221. Recently, solid polystywas increased to 104.2 ng by the ionophore. rene microcarriers were developed [ 14-161 For comparison, 7.9X IO6cells in monolayer with a negative surface charge and similar in composition to tissue culture plastic culture produced 48.6 ng 6-keto-PGFla. ware. Attachment of cells to the negatively The rates/cell for bead-bound cells and monolayer culture were therefore similar; charged surface probably occurs via amphoteric bridging proteins, such as plasma 6.1 and 5.2 ng/106 cells/l5 min respectively. In the case of the bead bed, however, the CIg. Therefore, preincubation of the beads Exp Cd Res 134 (1981)

Microcarrier culture of endothelium with serum greatly enhanced the subsequent attachment of endothelial cells. Most of our studies were conducted in static culture. We were surprised to observe the efficient colonization of beads without flow because in the literature concerning large-scale systems [23, 241emphasis has been placed upon concentration gradient effects and the careful design of stirring systems. It appears that in the small scale research systems such as described here, concentration gradient effects can be minimized either by distributing the beads in a monolayer or setting up adequate perfusion flow. Satisfactory final cell densities were obtained from a wide range of inoculation densities. The attainment of an average bead coverage approaching that of the maximal predicted density in static culture was most likely attributable to the absence of shear forces and collisions inherent in a stirred bead suspension. Recently Mered et al. [23] described a small-scale stirred system for the microcarrier culture of monkey kidney cells and primary chicken embryo cells on DEAE-Sephadex gel beads at low anion exchange capacity. They demonstrated an inverse relationship between bead concentration and final cell density and were unable to achieve a final cell density greater than 50% of that measured in plastic flasks. On the other hand, Levine et al. [21] in a similar system using human diploid cells (HEL299) achieved the same saturation density, irrespective of the initial cell inoculum. Using Vero cells, Mered et al. reported that the smallest inoculum (1 x 104/cm” growth surface) was the most effective, resulting in a 21-fold increase in cell population. It was not clear, however, whether lower inocula were tried. As described here, the lowest effective inoculum of endothelium was 3~ 103/cm2 growth surface

375

(about 3.5 cells/bead) which resulted in approx. a 50-fold increase in cell numbers in 11 days. Inocula less than this in the absence of FGF were less effective, the growth curves reaching a plateau well below confluent bead density (not shown). It appears that the growth of cells on sparsely populated beads was more successful in the presence of a number of beads which were already heavily populated. When naked beads were introduced into a microcarrier system containing confluent endothelium, they became colonized. These experiments show the feasibility of continuous culture of vascular endothelium. Such a system would be of great usefulness for endothelia which do not retain morphological and functional differentiation with multiple passages in culture. The maintenance of bead-associated endothelium in a perfusion system offers possibilities for the harvest of endothelialspecific molecules synthesized by, or modified within, the cells [25] and for the shortterm collection of prostaglandins synthesized by endothelium, particularly those with short half-lives [19, 201. The Biosilon beads are solid plastic; therefore the medium into which endothelial products may diffuse or be secreted comprises a small volume of the total bead bed. Furthermore the non-porous nature of the beads eliminates a large internal surface which might adsorb locally produced metabolites. Thus the stimulation of production of metabolites from a very large number of cells into a small volume of perfusate, as demonstrated for prostacyclin in this study, appears feasible. Other applications of the microcarrier system in cellular research include studies of cell interactions in which the introduction of microcarrier-associated cells in relatively large quantities into the environment Exp Ceil Res 134 (1981)

376

P. F. Davies

of a second cell type may offer advantages over conventional cocultivation. Alternatively, slow perfusion of a microcarrier bed may provide continuous freshly-conditioned medium piped directly to a bioassay system. Such applications are particularly relevant in vascular research in studies of the interactions between endothelium and vascular smooth muscle cells [25-271. The excellent technical assistance of Cathy Kerr, Sheila Cruise and Lesley Kuczera is gratefully acknowledged. I thank Dr Andrew Schafer for the radioimmunoassay of 6-Keto-PGFlo. The work was supported by USPHS NIH grant HL24612 and by BRSG grant SO7 RR05489 awarded bv the Biomedical Research Sunuort Grant Program. NIH.

9. Davies, P F, Selden, S C & Schwartz, S M, J cell

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(1980) 808. 17. Vogel, A, Raines, E, Kariya, B, Rivest, M-J &

Ross, R, Proc natl acad sci US 75 (1978) 2810. 18. Czervionke, R L, Smith, J B, Hoak, J C, Fry, G L & Haycroft, D S, Thromb res 14 (1979) 781. 19. Weksler, B B, Ley, C W & Jaffe, E A, J clin invest 62(1978) 923. 20. Baenziger, N L, Becherer, P R & Majerus, P W,

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Received December 22, 1980 Revised version received March 12, 1981 Accepted March 13, 1981

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