Optimization of Cell Culture Systems that Support Growth or Differentiation of
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.
Construction of a Cell System for Rapid Growth of EC
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
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
Construction of a Cell System for EC Monolayers that Exhibit Barrier Function
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
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
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
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:
- 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.
- 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).
- 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
- 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).
- 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-
- 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.
- 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).
- Use of this differentiation environment results in the formation of an intact EC
monolayer more rapidly than standard culture systems.
- Rubin, L.L., et al., J. Cell Biol. 115:1725 (1993).
- Huang, A.J., et al., J. Cell Biol. 120:1371 (1993).
- Hakkert, B.C., et al., Blood 78:2721 (1991).
- Morzycki, W., et al., Immunol. Lett. 25:331