Optimization Of Cell Culture Systems That Support Growth Or Differentiation Of Endothelial Cells

Optimization of Cell Culture Systems that Support Growth or Differentiation of Endothelial Cells

Introduction

Extracellular Matrices (ECM), growth factors, and cells all interact to define the cellular microenvironment. These interactions play a crucial role in regulating cellular physiology both in vivo and in vitro. In an effort to construct in vitro environments providing the optimal conditions for endothelial cells (EC), various combinations of ECM and growth factors were examined for their abilities to induce either cell growth or differentiation. EC from several sources (species and vessel types) were seeded into standard tissue culture flasks, or onto collagen I, fibronectin, or laminin, and were grown in a low-serum medium containing various growth factors including bFGF, EGF, or ECGS/ECGF. Cells grown on collagen I for five to seven days demonstrated a two- to four-fold increase in cell number compared to those seeded onto plastic, laminin, or fibronectin. The mitogenic effect of collagen I was maximal when EGF, ECGS, and heparin were included in the optimized low-serum medium. For induction of differentiation, however, the optimal ECM was either fibronectin or BD Matrigel™ Matrix, a reconstituted basement membrane. Optimization of key components of the microenvironment in vitro, such as the ECM, growth factors, and other soluble factors thus help to increase the rate of cellular growth and enhance the level of differentiation.

Results

These results indicate that the combination of hydrocortisone, EGF, ECGS, heparin (heparin sulfate proteoglycan gave similar results), and 2% FBS out-performed all other media tested. The medium formation (BD™ Endothelial Culture Medium) thus provides an optimized environment to promote rapid growth of EC under low-serum conditions.

These results indicate that collagen I is the optimal ECM promoting rapid proliferation of EC, supporting previous findings suggesting a mitogenic effect of collagen I for EC.¹ Furthermore, the results indicate that the combination of type I collagen and the optimized BD Endothelial Culture Medium (forming the BD BioCoat™ Endothelial Cell Growth Environment) supports rapid growth of EC better than all other ECM and medium combinations tested.

These results indicate the BD BioCoat™ Endothelial Cell Growth Environment provides all types of EC tested with culture conditions that optimally support their growth. In addition, this culture system allows for more rapid growth of EC than do standard culture conditions.

EC grown in the BD BioCoat™ Endothelial Cell Growth Environment may be used in a variety of applications. Some of these applications, however, may require intact EC monolayers exhibiting barrier function (e.g., studies of transendothelial leukocyte traffic or drug transport). BD Biosciences, therefore, sought to construct and optimize an in vitro environment promoting EC differentiation and the formation of a physiological barrier.

EC grown on permeable membranes with fibronectin, but not on those with collagen I, formed confluent monolayers demonstrating barrier function (deter- mined by measurement of transendothelial electrical resistance [TEER] and dye diffusion), and displayed markers of EC differentiation (vWF expression; AcLDL binding). EC seeded onto BD Matrigel Matrix, however, underwent tubulogenesis and generated capillary-like networks, simulating the process of angiogenesis. Monolayers of differentiated EC formed on microporous membranes with fibronectin proved useful in a variety of applications, including the study of transendothelial leukocyte traffic.

Figure 6. To examine the influence of the ECM on EC monolayer formation at the ultrastructural level, HUVECs were seeded (as described in Figure 5) onto BD Falcon™ Cell Culture Inserts without matrix or BD BioCoat™ Cell Culture Inserts with collagen I or fibronectin. After 48 hours, monolayers were fixed with glutaraldehyde and OsO₄ , and processed for examination by SEM.

SEM examination of these cultures yielded several observations:

1. After two days, HUVECs cultured on microporous membranes without ECM (A, B) demonstrated both flattened and rounder morphologies, indicating suboptimal culture conditions. In addition, large open areas were seen on the insert membrane (arrowheads), indicating lack of confluency.
2. After two days, HUVECs cultured on microporous membranes with type I collagen (C, D) were flatter than on membranes without ECM (C), but a number of cells were observed to be forming sprouts (C, D, and arrowheads) and to be migrating through the pores of the membranes (D). These monolayers were not confluent either, as indicated by the open areas of membrane visible between cells (C and arrowheads).
3. On microporous membranes with fibronectin (E, F), HUVECs were found to be completely flattened and demonstrated close intercellular junctions (E and arrowheads); when viewed more obliquely (F), the cells appear to form a confluent monolayer with a typical cobblestone morphology, and no open areas on the membrane.

Together (Figures 5 and 6), these results indicate that fibronectin is the optimal matrix for the rapid induction of intact monolayers of EC, while cells grown on membranes without ECM or with type I collagen do not form intact monolayers over this time course.

These results indicate that HUVEC monolayers grown on fibronectin microporous membranes form an intact barrier, as determined by TEER measurements, in agreement with those previously reported using standard culture conditions and much longer culture periods.² In agreement with microscopy analysis, TEER values for EC cultured on other ECMs or without ECM after 48 hours were lower, indicating a less intact (and thus more permeable) monolayer (Figures 5 and 6).

Having constructed an in vitro environment resulting in the rapid formation of intact EC monolayers demonstrating barrier function, the utility of this system in studies of transendothelial leukocyte trafficking was examined. Human neutrophils, lymphocytes, and monocytes were isolated from peripheral blood and seeded at 1.5 x 10⁶ leukocytes/insert onto intact EC monolayers on BD BioCoat™ Fibronectin Cell Culture Inserts (3 µm), and were incubated for 30 minutes to allow leukocytes to attach to and migrate through EC in response to various inducers. EC monolayers were then processed for SEM analysis by glutaraldehyde and OsO4 fixation, or were rinsed to remove nonmigrating cells and the number of migrating cells expressed as a percentage of the number of leukocytes seeded. Results represent means of three experiments.

Together, these results indicate that the culture system optimized to promote formation of intact EC monolayers can be used successfully in studies of transendothelial leukocyte trafficking. In addition, the monolayers obtained with two-day EC cultures are similar to those reported for standard culture systems that require longer culture periods for the establishment of intact EC monolayers.^2-4

Conclusions

1. Rapid growth of EC from a variety of sources can be achieved using an optimized combination of growth medium and a type I collagen substrate (the BD BioCoat Endothelial Cell Growth Environment).
2. Use of this growth environment results in more rapid growth of EC than use of traditional culture conditions (e.g., standard culture flasks, high serum- containing media).
3. While collagen I is the optimal ECM for promotion of EC growth, fibronectin is the matrix of choice for growth of intact monolayers of differentiated EC demonstrating barrier function.
4. Rapid differentiated function of endothelial cell monolayers can be achieved using an optimized culture medium and microporous membranes (3 µm) with fibronectin; the EC monolayers may be used to examine transendothelial migration of various types of leukocytes, and may find use in other applications (e.g., transendothelial drug transport).
5. Use of this differentiation environment results in the formation of an intact EC monolayer more rapidly than standard culture systems.

References

1. Rubin, L.L., et al., J. Cell Biol. 115:1725 (1993).
2. Huang, A.J., et al., J. Cell Biol. 120:1371 (1993).
3. Hakkert, B.C., et al., Blood 78:2721 (1991).
4. Morzycki, W., et al., Immunol. Lett. 25:331 (1990).